MicroRNAs Sensitize Cancers to Therapy

ABSTRACT

The present invention concerns methods and compositions regarding one or more microRNAs or variants thereof that are provided to an individual for a variety of medical treatments, including sensitization to cancer therapy or prevention of a cancer to become sensitized to a cancer therapy. In specific embodiments, the microRNAs include miR-520a (including at least miR-520a-3p and miR-520-5p), miR-520g, miR-520h, and functional variants thereof. In some embodiments, the cancer is ovarian cancer, and in particular embodiments, the cancer therapy is platinum-based chemotherapy.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/413,894 filed Jan. 9, 2015, which is a national phase applicationunder 35 U.S.C. §371 that claims priority to International ApplicationNo. PCT/US2013/050248 filed Jul. 12, 2013, which claims the benefit ofpriority to U.S. Provisional Patent Application Ser. No. 61/670,774,filed Jul. 12, 2012, and U.S. Provisional Patent Application Ser. No.61/775,498, filed Mar. 9, 2013, all of which applications areincorporated herein by reference in their entirety.

TECHNICAL FIELD

The field of the invention concerns at least compositions and methodsrelated to medical treatment, including at least treatment for cancer.In at least particular cases, the field of the invention concernssensitization of one or more cancers to a particular treatment. Incertain embodiments, the invention concerns at least cell biology andmolecular biology.

BACKGROUND OF THE INVENTION

Recent evidence has implicated small RNA transcripts known as microRNAsin at least certain types of cancer. MicroRNAs (miRNAs) are small,non-coding RNA transcripts that play a critical role in silencingpatterns of gene expression. The target specificity of individualmicroRNAs may be determined by a stretch of 6 nucleotides located from 2through 7 of the mature microRNA transcript. These nucleotides, known asthe “seed sequence” promote the binding of individual microRNAs to the3′ untranslated region of mRNA transcripts. The resulting duplex isincorporated into the RNA-induced silencing complex (RISC), leading totranslational repression often as a result of mRNA degradation. Becausesequences complementary to an individual microRNA seed can be found inmany different 3′UTRs, a single microRNA can target hundreds ofdifferent mRNAs across and within multiple pathways.

One exemplary type of cancer in which miRNAs may play a role is ovariancancer. Platinum-based chemotherapy is standard of care for all womennewly diagnosed with an epithelial ovarian cancer. These drugs includecarboplatin, cisplatin, oxaloplatin and may be used singly or incombination with other agents including paclitaxel, doxetaxel,gemcitabine, liposomal doxorubicin, bevacizumab, cyclophosphamide ortopotecan. Despite excellent response rates (>80%), there are a numberof issues associated with the use of platinum-based agents to treatovarian cancer. First, not all ovarian cancers respond well to thesetreatments. Approximately 20% of women with advanced ovarian cancersdemonstrate de novo resistance to these agents. Second, small(microscopic) volume disease persists after standard of care treatmenteven in those women who achieve a complete clinical response, as judgedby imaging, serum levels of tumor marker and physical examination, forexample. These implants eventually activate to repopulate diseaserecurrences. Third, recurrent ovarian cancer eventually becomesresistant to platinum-based therapy. Because platinum is currently byfar the most effective agent used to treat ovarian cancer, nearly allwomen with recurrent disease are treated with platinum either alone orin combination with some other agent. However, with continued use,almost all ovarian cancers become resistant to its cytotoxic activity.Ultimately, nearly all (>90%) of women with ovarian cancer die from aplatinum-resistant recurrence of their disease. Although higher doses ofplatinum agents have been shown to induce better responses, their use islimited because of the toxicity associated with dose escalation.Toxicities are also a problem for women being treated with currentstandard of care regimens. As many as 40% of women being treated forovarian cancer with current regimens experience problems with severeneutropenia, neuropathy and/or kidney damage that require dosereductions, further limiting the ability of clinicians to utilize doseintense or dose escalation strategies for the treatment of ovarian andother human cancers with platinum agents. Other side effects that canrequire dose reductions that limit efficacy include neutropenia, anemia,renal dysfunction, poor appetite, myalgias, fatigue, nausea andvomiting. In situations where these side effects limit the use ofplatinum agents or where platinum agents are no longer effective, anumber of phase II clinical trials have established the efficacy ofagents that are used by clinicians to manage this disease. These agentsinclude liposomal docetaxel, etoposide, gemcitabine, liposomaldoxorubicin, topotecan, bevacizumab, altretamine capecitabine,cyclophosphamide, irinotecan, malphalan, oxaliplatin, pemetrexed andvinolrelbine. (1-7). The efficacy of each of these agents has beentested in phase II clinical trials that support their use in the contextof platinum-resistant ovarian cancer. In addition to the dose regimensdocumented formally as part of phase II clinical trials, more recentdata indicates that alternative dosing regimens of these agents alone orin combination may enhance the efficacy. For example, recent evidencesuggests that the combination of paclitaxel administered on a weeklyschedule in combination with bevacizumab administered every two weekscan induce response rates as high as 70% (8, 9). It should be noted thatthis combination was tested using a study group that include women withboth platinum-sensitive and resistant ovarian cancer recurrences.Nonetheless, this observation is significant because response rates formost single agent regimens against platinum-resistant ovarian cancerrange from 12-30% (1-7). Because nearly all agents used to treat ovariancancer ultimately fall in their ability to arrest or reverse diseaseprogression. Survival for women with platinum resistant ovarian canceris 12-24 months.

The present invention addresses these issues and provides a solution fora long-felt need in the art to provide effective ovarian cancertreatment.

BRIEF SUMMARY OF THE INVENTION

The present invention is directed to methods and compositions related totreating or enhancing treatment of one or more medical conditions. Themedical conditions may be of any kind, but in specific embodiments themedical conditions may be for hyperproliferative disease. In someembodiments the medical conditions encompass cancer of any kind,preeclampsia, placental dysfunction in pregnancy, conditions involvingabnormal tissue differentiation such as colonic or endometrial polyps,preinvasive lesions such as cervical dysplasia and/or treating scars. Insome cases, stem cells are targeted with compositions of the invention,and such targeting may be for any purpose including, at least, targetingto reduce or inhibit the ability to repopulate tumors in vivo and evadechemotherapy treatments.

In particular embodiments, the individual has been diagnosed with cancerand is in need of cancer treatment. The individual may or may not haveone or more symptoms of cancer. The individual may be at risk for havingcancer. The individual may be at risk for being resistant to one or morecancer treatments, and that risk may or may not have been recognized bya medical provider.

In particular embodiments there are methods and compositions forenhancement of cancer treatment, including at least chemotherapy,immunotherapy, radiation therapy, and/or hormone therapy, for example.In some embodiments, the methods and compositions concern thesensitization of cancer to one or more treatments. The cancer may haveshown sensitization immediately upon treatment or the sensitization mayhave occurred after treatment had been provided for some time. Inparticular aspects, the sensitization to the treatment occurs directlyor indirectly through administration of one or more microRNAs to anindividual having received, receiving or who will be receiving cancertreatment. In specific embodiments, microRNAs 520 a, g, and/or hsensitize cancers to chemotherapy (including epithelial ovarian cancers,for example, to chemotherapy), immunotherapy, radiation therapy and/orhormone therapy.

In embodiments of the invention, one or more microRNAs of the inventionare provided to an individual for sensitizing a cancer in the individualto a cancer treatment. In some embodiments, the individual is diagnosedas having cancer resistant to therapy and is in need of treating thecancer such that it becomes sensitive to one or more therapies to whichit was previously resistant and is given the microRNA(s) with theexplicit purpose of sensitizing the cancer. In specific aspects, thecancer treatment targets an entity (such as a gene or gene product) thatis directly or indirectly affected by the microRNA of the invention. Insome embodiments, the microRNA impacts cell cycle checkpoints, DNAdamage repair pathways, and/or apoptosis, for example.

In some embodiments, microRNAs 520 a, g, and/or h prevent sensitizationof one or more cancers before they become resistant to a therapy. Inspecific embodiments, microRNAs 520a, g, and/or h reverse acquiredresistance to chemotherapy, including, for example, platinum, whichtypically occurs with time and can be because of genetic drift orselection of drug-resistant tumors with treatment, for example.

Although any treatment for any cancer may be affected by use of themicroRNAs of the invention, in specific embodiments the cancer isovarian cancer. The individual may have any type of ovarian cancer,including ovarian cancer types such as epithelial ovarian tumors(derived from the cells on the surface of the ovary); germ cell ovariantumors (derived from the egg producing cells within the body of theovary); and sex cord stromal ovarian tumors. The therapy for the ovariancancer may be for treating any stage of ovarian cancer, including stageI, II, III, or IV. The ovarian cancer may have been diagnosed byphysical exam, pelvic exam, blood tests, biopsy, and/or ultrasound, forexample. The individual may have been asymptomatic or may have hadsymptoms such as pressure or pain in the abdomen, pelvis, back, or legs;a swollen or bloated abdomen; nausea, indigestion, gas, constipation, ordiarrhea; and/or fatigue, for example. The ovarian cancer may or may notbe metastatic at the time of treatment with the microRNAs of theinvention. In specific embodiments the cancer treatment for which thecancer needs to be sensitized comprises platinum-based drugs, such ascarboplatin or cisplatin, with a taxane such as paclitaxel (Taxol) ordocetaxel (Taxotere). oxaliplatin, etoposide, ifosfamide, topotecan,gemcitabine, pegylated liposomal doxorubicin, doxorubicin,cyclophosphamide, epirubicin, altretamine irinotecan, pemetrexate,vinorelbine, tamoxifen, leuprolide, as well as other targeted biologicagents that have biologic activity against ovarian cancer. Examples ofthis latter category include bevacizumab, sunitinib and others.

In some embodiments, there is a method of sensitizing a cancer in anindividual to a cancer treatment, comprising the step of administeringto the individual an effective amount of a composition as follows: a) aRNA polynucleotide comprising SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3;or b) a RNA polynucleotide comprising sequence having one or more (forexample, one, two, three, or more) nucleotide alterations in SEQ IDNO:1, SEQ ID NO:2, or SEQ ID NO:3, wherein upon administering thecomposition to the individual, the cancer is thereby sensitized to thetreatment. In a specific embodiment, the composition is delivered to theindividual by liposome, nanosphere, nanoparticle, nanodiamonds,impregnanted polymer, multistage nanoparticles and/or gels. In somecases, the composition is delivered to the individual intravenously orintraperitoneally. In specific embodiments, the cancer is ovariancancer, uterine cancer, cervical cancer, lung cancer, colon cancer,breast cancer, or testicular cancer. In some aspects, the treatment ischemotherapy, immunotherapy, hormone therapy or a combination thereof.In particular cases, the treatment is platinum-based chemotherapy. Insome embodiments, the method further comprises delivering the cancertreatment to the individual. The composition may be delivered to theindividual prior to the cancer treatment or subsequent to the cancertreatment or concomitantly with the cancer treatment.

In some embodiments, there is an isolated composition, comprising an RNApolynucleotide having sequence comprising one or more (for example, one,two, three, or more) nucleotide alterations compared to SEQ ID NO:1, SEQID NO:2, or SEQ ID NO:3. In specific embodiments, the composition iscomprised in a pharmaceutically acceptable carrier.

In particular embodiments, there is a recombinant polynucleotidecomprising SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or a functional variantthereof. In specific embodiments, the expression of SEQ ID NO:1, SEQ IDNO:2, or SEQ ID NO:3 is under the control of an inducible promoter, suchas one selected from the group consisting of cytomegalovirus (CMV), Roussarcoma virus (RSV), human serum albumin (SA), a-1 antitrypsin (AAT),cytochrome P450 CYP1A2, CYP2C9, CYP2C18, CYP2D6, CYP3A4, amyloidprecursor protein (APP), nuclear factor j B (NFjB), and heat shockprotein 70. In some cases, there is an isolated cell comprising apolynucleotide of the invention.

In some embodiments, there is a kit for treating cancer in anindividual, said kit comprising in suitable container means: a) an RNApolynucleotide comprising SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3;and/or b) an RNA polynucleotide having sequence comprising one or more(for example, one, two, three, or more) nucleotide alterations comparedto SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3. In specific embodiments,the method further comprises one or more cancer treatments, such aschemotherapy, immunotherapy, and/or hormone therapy. In specific cases,the chemotherapy comprises platinum-based chemotherapy.

Altered patterns of miRNA expression have now been documented in manydifferent human diseases, including epithelial ovarian cancer (10-13).Recent evidence suggests that miRNA function may only becomebiologically evident under conditions of cellular stress (14, 15). Inone aspect, the set of microRNAs involved in the pathogenesis of thisdisease may actually be distinct from those microRNAs capable ofsensitizing formed tumors to current treatments. Because the majority ofwomen ultimately succumb to ovarian cancer, the microRNAs that couldpotentially sensitize these tumors to drugs such as cisplatin must beeither lost through deletion or remain repressed upon exposure totreatment. The inventors carried out a search for microRNAs that arepredicted to target multiple genes regulating cell cycle checkpoints andDNA damage pathways that have been selectively repressed through copylosses in the genome. A unique, primate-specific genomic locus at19q13.41 encoded more than 54 individual microRNAs. Although thisgenomic locus experiences frequent copy number variation in epithelialovarian cancers, the microRNAs it encodes are only infrequentlyexpressed in ovarian cancers. Nonetheless, these microRNAs arecollectively predicted to target both the G1-S and G2/M checkpoints aswell as multiple genes in the DNA damage pathways, such as ATM and ATR.On the basis of at least one or both of these two criteria, theinventors considered that microRNAs 520-a, g, and/or h are useful tosensitize epithelial ovarian cancers to established chemotherapies byaltering the expression of a large number of oncogenes known to becritical for regulating genomic stability, proliferation and apoptosis.The results provide insight into the mechanisms by which microRNAs canbe used to therapeutically target epithelial ovarian cancers andidentify a novel mechanism by which ovarian cancer can be sensitized tocancer therapy, including at least platinum-based chemotherapy.

In certain aspects to the invention, medical conditions other thancancer may be treated with methods and/or compositions of the invention.In particular the medical conditions are those in which administrationof microRNAs 520-a, g, and/or h are therapeutic. One particular exampleof such a medical condition includes at least preeclampsia. Anindividual that has preeclampsia or is suspected of having preeclampsia(symptoms include hypertension, proteinuria, and/or swelling) may beprovided with methods and/or compositions of the invention and, in somecases, they are given one or more other therapies, or Caesarean sectionor induction of labor (and therefore delivery of the placenta) mayoccur. The individual may have preeclampsia for any reason or at anystage during the pregnancy.

Another example of a medical condition in which administration ofmicroRNAs 520-a, g, and/or h is therapeutic includes placentaldysfunction in pregnancy. The dysfunction may be of any kind. In somecases, the placental dysfunction is placental insuffiency, whichincludes insufficient blood flow to the placenta and during pregnancy.Placental insufficiency includes symptoms such as amnion cell metaplasiaand placental thickness less than 1 cm, for example. It can also bereflected in impaired transplacental transport of nutrients. Theindividual being treated may be known to have placental dysfunction ormay be suspected of having placental dysfunction, and other treatmentsmay be given to the individual, such as bedrest, induction of labor, orCaesarean section.

An additional example of a medical condition in which administration ofmicroRNAs 520-a, g, and/or h is therapeutic includes treating scars. Thescar may be of any kind, including external or internal, and the causeof the scar may be of any kind. The scar may be anywhere on or in thebody and may be new or old and includes categories of hypertrophic scarsknown as keloids. This includes the formation or prevention ofintraperitoneal adhesions. In specific embodiments, the methods and/orcompositions of the invention are provided to the scar after itsformation or are alternatively or in addition provided to a wound sitebefore scar formation. Any type of scar may be treated including keloidscars, contracture scars, hypertrophic scars, and/or acne scars. Thecomposition of the invention may be provided to the individual locallyor systemically. The composition of the invention may be provided to theindividual with another therapeutic composition for scar treatmentand/or prevention (such as silicone or onion extract, for example).

The foregoing has outlined rather broadly the features and technicaladvantages of the present invention in order that the detaileddescription of the invention that follows may be better understood.Additional features and advantages of the invention will be describedhereinafter which form the subject of the claims of the invention. Itshould be appreciated by those skilled in the art that the conceptionand specific embodiment disclosed may be readily utilized as a basis formodifying or designing other structures for carrying out the samepurposes of the present invention. It should also be realized by thoseskilled in the art that such equivalent constructions do not depart fromthe spirit and scope of the invention as set forth in the appendedclaims. The novel features which are believed to be characteristic ofthe invention, both as to its organization and method of operation,together with further objects and advantages will be better understoodfrom the following description when considered in connection with theaccompanying figures. It is to be expressly understood, however, thateach of the figures is provided for the purpose of illustration anddescription only and is not intended as a definition of the limits ofthe present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the present invention, reference isnow made to the following descriptions taken in conjunction with theaccompanying drawing, in which:

FIGS. 1A-1C: Genomic copy number variation and microRNA expression inepithelial ovarian cancers. (FIG. 1A) Copy number gains (yellow) andlosses (blue) identified in a 500 specimens of high grade serous ovariancancers are plotted as a function of chromosome location. Location ofknown human microRNAs (mirBase v.16) are identified in gray. Note thatcopy number gains and losses at 19q13.41, the site of miR-520g/h occurfrequently in EOC at a unique genomic locus encoding more than 45 maturehuman microRNAs. (FIG. 1B) Expression of specific microRNAs in specimensof fallopian tube, short term primary culture of ovarian surfaceepithelia and epithelial ovarian cancers evaluated by Next GenerationSequencing of cloned small RNA libraries. Note that very few small RNAtranscripts could be detected in any of the specimens studied thatcorrespond to miR-520a, miR-520g/h. (FIG. 1C) Outcome demographicsindicate that survival is significantly improved when subset of ovariancancer patients with highest expression of miR-520g/h are compared to340 patients with lowest levels of miR-520g/h expression.

FIGS. 2A-2H. Impact of Specific microRNAs on Ovarian CancerProliferation and Apoptosis. MTS and Caspase Glo 3/7 assays were used tocompare rates of proliferation (FIGS. 2A, 2C, 2E, 2G) and apoptosis(FIGS. 2B, 2D, 2F, 2H) in OVCAR8 cells transiently transfected mimicsfor miR-520a-3p (FIGS. 2A, 2B), miR-520a-5p (FIGS. 2C, 2D), miR-520h(FIGS. 2E, 2F) and miR-1323 (FIGS. 2G, 2H) or non-silencing controls.Mir-520g/h significantly increased the proliferation (n=12, p<0.001),whereas miR-520a-3p (See 2B, n=12; p=0.008), miR520a-5p (See 2D; n=12;p=0.00 and miR-1323 (see 2H; n=12, p=0.0) decreased apoptosis whencompared to OVCAR8 cells transfected with non-silencing microRNA mimiccontrols transfected under identical conditions. Hairpin microRNAinhibitors were not studied as levels of endogenous 19q locus microRNAswere undetectable (data not shown).

FIG. 3. Ectopic Expression of miR-520h Sensitizes Established OvarianCancer Cell Lines to Chemotherapy. Proliferation and Apoptosis assaysfollowing acute transfection of both OVCAR8 and SKOV3ip1 cells withmiR-520h and miR-520 is shown. miR-520h significantly reduced rates ofproliferation while decreasing apoptosis as measured by commerciallyavailable Caspase 3/7 assays. Acute transfection of SKOV3ip1 and OVCAR8cells with synthetic mimic for miR-520a also resulted in decreased ratesof apoptosis but only decreased proliferation only in OVCAR8 cells. Nocells were exposed to cisplatin or other platinum-based chemotherapyagents in these studies. All differences are significant (p<0.05). Errorbars reflect standard deviation calculated from biologic replicates.

FIGS. 4A-4D. Viability of OVCAR8 and SKOV3ip1 cells stably expressingeither microRNA incubated with clinically relevant concentrations ofcisplatin. The dose response relationship for cisplatin was establishedfor OVCAR8 and SKOV3ip1 cells stably expressing either miR-520g/h,miR-520a or non-silencing microRNA mimic. The expression of eithermiR-520g/h or miR-520a sensitized OVCAR8 clones to concentrations ofcisplatin ≦5 μg/mL. This was associated with significantly lower ratesof apoptosis at all concentrations of cisplatin tested (up to 10 μg/mL).Both microRNAs also appeared to sensitize SKOV3ip1 cells, althoughmiR-520a appeared to have less impact in SKOV3ip1 than OVCAR8 cells. Alldifferences are significant with p<0.05. Error bars reflect standarddeviation calculated from 6 biologic replicates.

FIGS. 5A-5G. Overexpression of miR-520g/h alters the growth andmetastasis of ovarian cancer xenografts. OVCAR8 cells stably expressingboth RFP and either miR-520h (FIG. 5C), non-silencing mimic control(FIG. 5B) or empty vector (FIG. 5A) were injected eitherintraperitoneally or subcutaneously. All animals were followed for 6weeks using a whole animal fluorescent imaging system as described toimage xenografts in situ. FIG. 5D. Fluorescent imaging demonstrated alarge number of smaller implants in animals receiving OVCAR8 cellsoverexpressing miR-520g/h. (FIG. 5E) These results were confirmed atnecropsy when individual implant size was directly measured. (FIG. 5F)Total intraperitoneal tumor burden between the three experiments groupsdid not differ. (FIG. 5G) Individual implants were also observed whenOVCAR8 cells were subcutaneously injected. All differences aresignificant with p<0.05. Error bars reflect standard deviationcalculated from 6 biologic replicates.

FIGS. 6A-6G. Expression of miR-520g/h Improves Survival for XenograftedAnimals. Survival analysis for Fox1^(nu/nu) mice xenografted with2.5×10⁶ OVCAR8 cells expressing either mir-520g/h mimic, non-targetingmimic control or empty vector. Median survival for animals xenograftedwith miR-520g/h expressing cells is significantly longer (113 days) thananimals xenografted with cells expressing either non-targeting microRNAmimic (90 days) or empty vector (79 days). Cisplatin doses wereadministered as noted.

FIG. 7. Model for mir-520h Function in Epithelial Ovarian Cancer.Bioinformatic analysis predict that miR-520h is predicted to directlytarget multiple gene products involved in critical cell cyclecheckpoints (ATM, Cyclin B, Cyclin D, CHK1 and p53), DNA damage repairpathways (FANCD2, BRCA1), and apoptosis (FAS, FADD, Caspase 7).

FIGS. 8A-8H. Mir-520g/h expression silences the response of apoptosisand DNA damage repair pathways activated by cisplatin exposure. (FIG.8A) Expression of the tumor suppressor ATM is markedly reduced in OVCAR8cells stably expressing miR-520 when compared to clones stablytransfected with either non-silencing control or empty vector. (FIG.8B). MiR-520h promotes a proteolytic degradation of ATM that normallyoccurs in response to cisplatin. This is evidenced by increased morerobust expression of cleaved ATM dimers, the smaller protein product(ΔATM) recognized by the ATM antibody (see labeled arrow, FIG. 8B).Increased ATM degradation (FIG. 8C) is associated with decreased levelsof ATM and phosphoATM. Consistent with increased levels of ATM activity,increased levels of phopho-CHK2 (T68) are observed in the presence ofmiR-520h. In addition, expression of miR-520h blunts the responses ofmultiple gene products to cisplatin. These gene products include CHK1(FIG. 8D), CHK2 (FIG. 8E), CDC25C (FIG. 8F), CASP7 (FIG. 8G) and BRCA1(FIG. 8H). Note that the impact of increased ATM activity does notuniformly impact all ATM-regulated pathways, as decreased levels ofCDC25c and phospho-CDC25c protein are found in OVCAR8 cells (FIG. 8B),likely as a result of miR-520g/h-induced translational silencing of itsupstream regulator CHK1 when OVCAR8 cells are incubated with cisplatin(FIG. 8D). All qPCR results marked by an asterisk (*) significant withp<0.05. Error bars reflect standard deviation calculated from 5 biologicreplicates.

FIGS. 9A and 9B: Mimics for miR-520g/h impact creation of spheroids, akey intermediate in metastasis and cancer stem cells and impact patternsof cancer stemness. OVCAR8 and SVOK3ip1 cells stably expressing mimicsfor miR-520h demonstrate decreased capacity to form spheroids,multicellular aggregates that play a key role in ovarian cancermetastasis. (FIG. 9A) Spheroids have also been used to isolatesubpopulations of cells that fulfill many of the criteria of cancer stemcells and express enhanced levels of specific gene products includingEZH2, OCT4 and NANOG associated with pleuripotency in human embryonicstem cells (16). (FIG. 9B) Expression of miR-520gh impacts patterns ofand eliminates increased expression of specific patterns of geneexpression (i.e. lin28A/B) associated with stemness that are inducedwhen ovarian cancer cells (OVCAR8 and SKOV3ip1) are cultured in mediathat induce spheroid formation.

FIG. 10: miR-520h sensitizes cell lines derived from uterine cancer tocisplatin. UPSC Ark1 cells stably transfected with lentiviral vectordriving the expression of either miR-520h or a non-targeting miRNAcontrol were incubated with increasing concentrations of cisplatin. Asdemonstrated above, the IC50 for cisplatin was reduced by more than 90%in cells expressing miR-520h

DETAILED DESCRIPTION OF THE INVENTION

As used herein the specification, “a” or “an” may mean one or more. Asused herein in the claim(s), when used in conjunction with the word“comprising”, the words “a” or “an” may mean one or more than one. Asused herein “another” may mean at least a second or more. In specificembodiments, aspects of the invention may “consist essentially of” or“consist of” one or more sequences of the invention, for example. Someembodiments of the invention may consist of or consist essentially ofone or more elements, method steps, and/or methods of the invention. Itis contemplated that any method or composition described herein can beimplemented with respect to any other method or composition describedherein. Embodiments discussed in the context of methods and/orcompositions of the invention may be employed with respect to any othermethod or composition described herein. Thus, an embodiment pertainingto one method or composition may be applied to other methods andcompositions of the invention as well.

Throughout this application, the term “about” is used to indicate that avalue includes the standard deviation of error for the device or methodbeing employed to determine the value.

The term “sensitizes” as used herein refers to one or more agents thatpromote the efficacy or improve function of a cancer treatment.

I. General Embodiments of the Invention

Using a dataset of gene and micro/RNA expression generated from nearly500 high grade epithelial ovarian cancers, the inventors identified aunique genomic locus at 19q13.41 that encodes more than 45 individualmicroRNAs. Using Kaplan-Meier survival analyses, the inventors foundthat levels for microRNAs 520-a, g, and/or h encoded by this locuscorrelate with outcomes for women with ovarian cancer. In certainembodiments, the data indicate that mimics for at least one of thesemicroRNAs (miR-520h, for example) can be used to dramatically sensitizeestablished ovarian cancer cell lines to cisplatin both in vitro and invivo. The IC₅₀ for cisplatin in multiple ovarian cancer cell lines isreduced as much as 4-fold in OVCAR8 and SKOV3ip1 cells (p<0.001). At amolecular level and in certain embodiments, miR-520h accomplishes thisfeat by targeting multiple gene products previously implicated inplatinum responses. These include key components of DNA damage repairpathways (ATM), gene products regulating the G1-S and G2-M cell cyclecheckpoints, apoptosis and cell migration. Most dramatically, therepresentative miR-520h improves survival of mice xenografted withovarian cancer when treated with very low dose cisplatin at less than 5%of those currently used to clinically manage ovarian cancer in women.Mean survival for mice receiving miR-520h, for example, is 130 dayscompared to when mice are treated with a biweekly maintenance IV regimenof 5 mg/m² cisplatin (compared to 50-100 mg/m² typically used to achievetherapeutic responses.) These data provide dramatic and convincingevidence that miR-520h can be used to sensitize ovarian cancer toplatinum-based chemotherapy and improve survival. Other microRNAs from19q13.41 may be similarly used.

Treatment with one or more microRNAs of the present invention can beincorporated into frontline therapy for newly diagnosed epithelialovarian cancer (EOC). Given the widespread use of platinum-based agentsto treat other human cancers, a role for microRNA (including miR-520-a,g, and/or h: 5′-caaagug is the seed for miR-520g/h; the seed formiR-520a is 5′aagugcu) treatments can also be reasonably anticipated inat least angiosarcomas, lung cancer, cervical cancer, colon cancer,bladder cancer, testicular cancer, head and neck cancers, and so forth.Use of one or more microRNAs of the invention (including miR-520-a, g,and/or h) will improve outcomes for individuals diagnosed with awide-range of malignancies.

The compositions of the invention are unique in that they targetmultiple gene products across different signaling pathways involved inplatinum resistance. The fact that response to platinum chemotherapy isdetermined by multiple gene products is an aspect that has thwarted theeffective clinical development of agents for this purpose.

In embodiments of the invention, one can demonstrate in vivo safety andefficacy of liposomal miR-520h nanoparticles using xenograft models ofovarian cancer. In embodiments of the invention, one can determine theideal route, timing and dosage for miR-520h liposomes necessary toachieve optimal tumor shrinkage. In embodiments of the invention, onecan demonstrate that miR-520h liposomes administered at an optimaltiming and dose given with combination platinum-based chemotherapy atreduced dosage improve outcomes even when compared to combinationplatinum-based chemotherapy at standard doses.

In specific embodiments, the microRNA treatments are delivered ininfusion centers, clinics and hospital where ovarian cancer patientscurrently receive care.

In certain aspects of the invention, one or more microRNAs are employedto sensitize cancer to platinum-based chemotherapy agents that work bycross-linking subunits of DNA. These agents act during all parts of thecell cycle and impair DNA synthesis, transcription, and function. Firstgeneration platinum-based chemotherapeutics include cisplatin, althoughit is highly toxic and can severely damage the kidneys. The secondgeneration platinum-complex carboplatin(cis-diammine-[1,1-cyclobutanedicarboxylato]platinum(II)) much lesstoxic in comparison and have fewer kidney-related side effects.Oxaliplatin, which is third generation platinum-based complex, is usedto treat colon cancer, for example.

In certain embodiments of the invention, microRNAs miR-520-a, g, and/orh dramatically sensitize ovarian cancers to platinum-based chemotherapy.Synthetic mimics for miR-520h, for example, can be safely delivered towomen with ovarian cancer and used to increase the potency ofplatinum-based agents, while reducing the incidence of side effects anddramatically improving outcomes. The inventors envision the use ofliposomes containing miR-520h mimics (or those for mir-520a or g) thatcan be delivered either intraperitoneally or intravenously as part oftreatments for ovarian cancer in a number of clinical contexts. Thereagents used to synthesize these liposomes can allow for the costeffective, readily scalable mass production of a clinical vector thatcan be incorporated into standard of care treatments for ovarian cancer.Given the central role that platinum-based agents play in standard ofcare treatment for other human cancers, the present invention is usefuland effective for treating a wide range of other cancers.

II. Exemplary MicroRNAs of the Invention

In embodiments of the invention, microRNAs miRNA 520 a, g, and/or h areprovided to an individual for cancer treatment. In particularembodiments, one or more microRNAs are provided to an individual toimprove the efficacy of a treatment. The cancer treatment may be of anykind, but in specific cases the treatment is chemotherapy, radiationtherapy, hormone therapy, or immunotherapy or targeted therapy directedat specific subsets of cancer cancers, including cancer stem cells(CDCs) or cancer initiating cells (CICs), vascular cells, or stroma. Incertain aspects, the anti-cancer treatment is platinum-basedchemotherapy.

The term “miRNA” is used according to its ordinary and plain meaning andrefers to a microRNA molecule found in eukaryotes that is involved inRNA-based gene regulation. See, e.g., Carrington et ai., 2003, which ishereby incorporated by reference. The term may be used to refer to theRNA molecule processed from a precursor.

In certain aspects, the one or more microRNAs is selected from the groupconsisting of miR-520a (including at least miR-520a-3p and miR-520-5p),miR-520g, miR-520h, and functional variants thereof.

hsa-mir-520h MI0003175:

(SEQ ID NO: 1) UCCCAUGCUGUGACCCUCUAGAGGAAGCACUUUCUGUUUGUUGUCUGAGAAAAAACAAAGUGCUUCCCUUUAGAGUUACUGUUUGGGA

hsa-mir-520g MI0003166:

(SEQ ID NO: 2) UCCCAUGCUGUGACCCUCUAGAGGAAGCACUUUCUGUUUGUUGUCUGAGAAAAAACAAAGUGCUUCCCUUUAGAGUGUUACCGUUUGG GA

hsa-mir-520a MI0003149

(SEQ ID NO: 3) CUCAGGCUGUGACCCUCCAGAGGGAAGUACUUUCUGUUGUCUGAGAGAAAAGAAAGUGCUUCCCUUUGGACUGUUUCGGUUUGAG

It is well known in the art that modifications can be made to thesequence of a miRNA or a pre-miRNA without disrupting miRNA activity,including miR-520a (including at least miR-520a-3p and miR-520-5p),miR-520g, miR-520h, and functional variants thereof. As used herein, theterm “functional variant” of a miRNA sequence refers to anoligonucleotide sequence that varies from the natural miRNA sequence,but retains one or more functional characteristics of the miRNA (e.g.enhancement of cancer cell susceptibility to chemotherapeutic agents,cancer cell proliferation inhibition, induction of cancer cellapoptosis, specific miRNA target inhibition). In some embodiments, afunctional variant of a miRNA sequence retains all of the functionalcharacteristics of the miRNA. In certain embodiments, a functionalvariant of a miRNA has a nucleobase sequence that is a least about 60%,65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% identical to the miRNA or precursor thereof over a region of about5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 ormore nucleobases, or that the functional variant hybridizes to thecomplement of the miRNA or precursor thereof under stringenthybridization conditions. Accordingly, in certain embodiments thenucleobase sequence of a functional variant may be capable ofhybridizing to one or more target sequences of the miRNA. Alsoencompassed in the invention is the use of oligonucleotides of anylength that are processed into mature miRNAs, including miR-520a(including at least miR-520a-3p and miR-520-5p), miR-520g, miR-520h, andfunctional variants thereof. A functional variant may comprise one, two,three, four, five, six, seven, eight, or more alterations compared to aparticular miRNA or precursor thereof.

In some embodiments, the complementary strand is modified so that achemical group other than a phosphate or hydroxyl is at its 5′ terminus.The presence of the 5′ modification apparently eliminates uptake of thecomplementary strand and subsequently favors uptake of the active strandby the miRNA protein complex. The 5′ modification can be any of avariety of molecules known in the art, including NH₂, NHCOCH₃, andbiotin.

In another embodiment, the uptake of the complementary strand by themiRNA pathway is reduced by incorporating nucleotides with sugarmodifications in the first 2-6 nucleotides of the complementary strand.It should be noted that such sugar modifications can be combined withthe 5′ terminal modifications described above to further enhance miRNAactivities.

In some embodiments, the complementary strand is designed so thatnucleotides in the 3′ end of the complementary strand are notcomplementary to the active strand. This results in double-strand hybridRNAs that are stable at the 3′ end of the active strand but relativelyunstable at the 5′ end of the active strand. This difference instability enhances the uptake of the active strand by the miRNA pathway,while reducing uptake of the complementary strand, thereby enhancingmiRNA activity.

In some embodiments, miRNA sequences of the invention may be associatedwith a second RNA sequence that may be located on the same RNA moleculeor on a separate RNA molecule as the miRNA sequence. In such cases, themiRNA sequence may be referred to as the active strand, while theencoded RNA sequence, which is at least partially complementary to themiRNA sequence, may be referred to as the complementary strand. Theactive and complementary strands may be hybridized to generate adouble-stranded RNA that is similar to a naturally occurring miRNAprecursor. The activity of a miRNA may be optimized by maximizing uptakeof the active strand and minimizing uptake of the complementary strandby the miRNA protein complex that regulates gene translation. This canbe done through modification and/or design of the complementary strand,for example.

miRNA used in the reaction may be obtained by a variety of methods andfrom a variety of sources. The miRNA may be obtained from a biologicalsample, such as a cell, tissue, or organ. It may be isolated from abiological sample that contains other RNA molecules as well, such asmRNA, tRNA, and/or rRNA. In certain instances, total RNA is firstisolated from the sample and then the miRNA is separated from the otherRNA, thereby enriching for miRNA. In some embodiments, the miRNA hasbeen isolated away from other RNA to enrich for the miRNA, such that themiRNA substantially pure, meaning it is at least about 80%, 85%, 90%,95% pure or more, but less than 100% pure, with respect to other RNAmolecules. Alternatively, enrichment of miRNA may be expressed in termsof fold enrichment. In certain embodiments, miRNA is enriched by about,at least about, or at most about 5×, 10×, 20×, 30×, 40×, 50×, 60×, 70×,80×, 90×, 100×, 250×, 500×, 1000×, and so forth. In some embodiments,the miRNA polynucleotide is synthesized.

MicroRNA molecules (“miRNAs”) are generally 21 to 22 nucleotides inlength, though lengths of 19 and up to 23 nucleotides have beenreported. The miRNAs are each processed from a longer precursor RNAmolecule (“precursor miRNA”). Precursor miRNAs are transcribed fromnon-protein-encoding genes. The precursor miRNAs have two regions ofcomplementarity that enables them to form a stem-loop- or fold-back-likestructure, which is cleaved by an enzyme called Dicer in animals. Diceris ribonuclease III-like nuclease. The processed miRNA is typically aportion of the stem.

The processed miRNA (also referred to as “mature miRNA”) become part ofa large complex to down-regulate a particular target gene or genes.Examples of animal miRNAs include those that imperfectly basepair withthe target, which halts translation (Olsen et al, 1999; Seggerson et al,2002)

In some embodiments of the invention, there is an isolated composition,comprising SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, respectively,wherein certain nucleotides therein are non-variable and/or one or more(including 1, 2, 3, 4, 5, 6, 7, 8, 9, or more) of the remainingnucleotides in the sequence are variable compared to SEQ ID NO:1, SEQ IDNO:2, or SEQ ID NO:3, respectively, wherein the composition has activityof sensitizing cancer to a therapy.

In specific aspects, variants of the miRNA sequence are employed. Incertain embodiments, the seed sequence (which, for example, is sharedbetween miR-520h and miR-520g) is not varied in the variants of therespective miRNA sequence. In other aspects, the anchor sequence is notvaried in the variants of the respective miRNA sequence.

III. Inhibitors of miRNAs

In some embodiments of the invention, one or more inhibitors of miR-520a(including at least miR-520a-3p and miR-520-5p), miR-520g, miR-520h, andfunctional variants thereof, and/or methods of using them areencompassed in the invention. Methods of using inhibitors of miR-520 a,g, and/or h include those for treating an individual in need thereof. Incertain embodiments, the inhibitors of miR-520 a, g, and/or h areemployed in medical conditions in which there is an increased level ofmiR-520 a, g, and/or h compared to the levels in a normal standard. Themedical condition may be of any kind having such elevated levels, but inspecific embodiments the medical condition is angiosarcomas, conditionsthat rely on dysregulated pluripotency (i.e. stems cells).

The inhibitors of miR-520 a, g, and/or h encompassed in the inventionmay be of any kind, but in specific embodiments the inhibitors compriseat least nucleic acid, protein, and/or small molecules. In specificembodiments the inhibitors are nucleic acid that have sequence that iscomplementary to at least part of a miR-520 a, g, and/or h. Inparticular embodiments the inhibitor is an antisense inhibitor. In atleast some cases, the inhibitor is specific for the respective miR-520a, g, and/or h. Design of the nucleic acid inhibitor takes intoconsideration AT-rich microRNA targets, if applicable. Commercialproducts and assays for using them are available to the skilled artisan(for example, Exiqon, Woburn, Mass.; and Life Technologies TaqMan®MicroRNA Assays; ThermoScientific, Waltham, Mass.).

IV. Nucleic Acids

The present invention concerns miRNAs that can be used in therapeuticapplications. The RNA may have been endogenously produced by a cell, orbeen synthesized or produced chemically or recombinantly. They may beisolated and/or purified. The term “miRNA,” unless otherwise indicated,refers to the processed RNA, after it has been cleaved from itsprecursor.

Nucleic acids of the invention may have regions of identity orcomplementarity to another nucleic acid. It is contemplated that theregion of complementarity or identity can be at least 5 contiguousresidues, though it is specifically contemplated that the region is, isat least, or is at most 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, or more contiguous nucleotides, whereappropriate. It is further understood that the length of complementaritybetween an miRNA and its target gene(s) are such lengths. Moreover, thecomplementarity may be expressed as a percentage, meaning that thecomplementarity between a miRNA and its target is 80% or greater overthe length of the miRNA or a noted fragment thereof. On someembodiments, complementarity is or is at least 80%, 85%, 90%, 91%, 92%,95%, 97%, 99%, or 100%, for example.

It is understood that an miRNA can be derived from genomic sequences ora gene. In this respect, the term “gene” is used for simplicity to referto the genomic sequence encoding the precursor miRNA for a given miRNA.However, embodiments of the invention may involve genomic sequences of amiRNA that are involved in its expression, such as a promoter or otherregulatory sequences.

The term “recombinant” may be used and this generally refers to amolecule that has been manipulated in vitro or that is the replicated orexpressed product of such a molecule.

The term “nucleic acid” is well known in the art. A “nucleic acid” asused herein will generally refer to a molecule (one or more strands) ofDNA, RNA or a derivative or analog thereof, comprising a nucleobase. Anucleobase includes, for example, a naturally occurring purine orpyrimidine base found in DNA (e.g., an adenine “A,” a guanine “G,” athymine “T” or a cytosine “C”) or RNA (e.g., an A, a G, an uracil “U” ora C). The term “nucleic acid” encompasses the terms “oligonucleotide”and “polynucleotide,” each as a subgenus of the term “nucleic acid.”

As used herein, “hybridization”, “hybridizes” or “capable ofhybridizing” is understood to mean the forming of a double or triplestranded molecule or a molecule with partial double or triple strandednature. The term “anneal” as used herein is synonymous with “hybridize.”The term “hybridization”, “hybridize(s)” or “capable of hybridizing”encompasses the terms “stringent condition(s)” or “high stringency” andthe terms “low stringency” or “low stringency condition(s).”

As used herein “stringent condition(s)” or “high stringency” are thoseconditions that allow hybridization between or within one or morenucleic acid strand(s) containing complementary sequence(s), butprecludes hybridization of random sequences. Stringent conditionstolerate little, if any, mismatch between a nucleic acid and a targetstrand. Such conditions are well known to those of ordinary skill in theart, and are preferred for applications requiring high selectivity.Non-limiting applications include isolating a nucleic acid, such as agene or a nucleic acid segment thereof, or detecting at least onespecific mKNA transcript or a nucleic acid segment thereof, and thelike.

Stringent conditions may comprise low salt and/or high temperatureconditions, such as provided by about 0.02 M to about 0.5 M NaCl attemperatures of about 42° C. to about 70° C. It is understood that thetemperature and ionic strength of a desired stringency are determined inpart by the length of the particular nucleic acid(s), the length andnucleobase content of the target sequence(s), the charge composition ofthe nucleic acid(s), and to the presence or concentration of formamide,tetramethylammonium chloride or other solvent(s) in a hybridizationmixture.

It is also understood that these ranges, compositions and conditions forhybridization are mentioned by way of non-limiting examples only, andthat the desired stringency for a particular hybridization reaction isoften determined empirically by comparison to one or more positive ornegative controls. Depending on the application envisioned it ispreferred to employ varying conditions of hybridization to achievevarying degrees of selectivity of a nucleic acid towards a targetsequence. In a non-limiting example, identification or isolation of arelated target nucleic acid that does not hybridize to a nucleic acidunder stringent conditions may be achieved by hybridization at lowtemperature and/or high ionic strength. Such conditions are termed “lowstringency” or “low stringency conditions”, and non-limiting examples oflow stringency include hybridization performed at about 0.15 M to about0.9 M NaCl at a temperature range of about 20° C. to about 50° C. Ofcourse, it is within the skill of one in the art to further modify thelow or high stringency conditions to suite a particular application.

A. Nucleobases

As used herein a “nucleobase” refers to a heterocyclic base, such as forexample a naturally occurring nucleobase (i.e., an A, T, G, C or U)found in at least one naturally occurring nucleic acid (i.e., DNA andRNA) and naturally or non-naturally occurring derivative(s) and analogsof such a nucleobase. A nucleobase generally can form one or morehydrogen bonds (“anneal” or “hybridize”) with at least one naturallyoccurring nucleobase in manner that may substitute for naturallyoccurring nucleobase pairing (e.g., the hydrogen bonding between A andT, G and C, and A and U).

“Purine” and/or “pyrimidine” nucleobase(s) encompass naturally occurringpurine and/or pyrimidine nucleobases and also derivative(s) andanalog(s) thereof, including but not limited to, those a purine orpyrimidine substituted by one or more of an alkyl, caboxyalkyl, amino,hydroxyl, halogen (i.e., fluoro, chloro, bromo, or iodo), thiol oralkylthiol moiety. Preferred alkyl (e.g., alkyl, caboxyalkyl, etc.)moieties comprise of from about 1, about 2, about 3, about 4, about 5,to about 6 carbon atoms. Other non-limiting examples of a purine orpyrimidine include a deazapurine, a 2,6-diaminopurine, a 5-fluorouracil,a xanthine, a hypoxanthine, a 8-bromoguanine, a 8-chloroguanine, abromothymine, a 8-aminoguanine, a 8-hydroxyguanine, a 8-methylguanine, a8-thioguanine, an azaguanine, a 2-aminopurine, a 5-ethylcytosine, a5-methylcyosine, a 5-bromouracil, a 5-ethyluracil, a 5-iodouracil, a5-chlorouracil, a 5-propyluracil, a thiouracil, a 2-methyladenine, amethylthioadenine, a N5N-diemethyladenine, an azaadenines, a8-bromoadenine, a 8-hydroxyadenine, a 6-hydroxyaminopurine, a6-thiopurine, a 4-(6-aminohexyl/cytosine)5 and the like. Other examplesare well known to those of skill in the art.

A nucleobase may be comprised in a nucleoside or nucleotide, using anychemical or natural synthesis method described herein or known to one ofordinary skill in the art. Such nucleobase may be labeled or it may bepart of a molecule that is labeled and contains the nucleobase.

B. Nucleosides

As used herein, a “nucleoside” refers to an individual chemical unitcomprising a nucleobase covalently attached to a nucleobase linkermoiety. A non-limiting example of a “nucleobase linker moiety” is asugar comprising 5-carbon atoms (i.e., a “5-carbon sugar”), includingbut not limited to a deoxyribose, a ribose, an arabinose, or aderivative or an analog of a 5-carbon sugar. Non-limiting examples of aderivative or an analog of a 5-carbon sugar include a2′-fluoro-2′-deoxyribose or a carbocyclic sugar where a carbon issubstituted for an oxygen atom in the sugar ring.

Different types of covalent attachment(s) of a nucleobase to anucleobase linker moiety are known in the art. By way of non-limitingexample, a nucleoside comprising a purine (i.e., A or G) or a7-deazapurine nucleobase typically covalently attaches the 9 position ofa purine or a 7-deazapurine to the 1′-position of a 5-carbon sugar. Inanother non-limiting example, a nucleoside comprising a pyrimidinenucleobase (i.e., C, T or U) typically covalently attaches a 1 positionof a pyrimidine to a 1′-position of a 5-carbon sugar (Kornberg andBaker, 1992).

C. Nucleotides

As used herein, a “nucleotide” refers to a nucleoside further comprisinga “backbone moiety”. A backbone moiety generally covalently attaches anucleotide to another molecule comprising a nucleotide, or to anothernucleotide to form a nucleic acid. The “backbone moiety” in naturallyoccurring nucleotides typically comprises a phosphorus moiety, which iscovalently attached to a 5-carbon sugar.

The attachment of the backbone moiety typically occurs at either the 3′-or 5′-position of the 5-carbon sugar However, other types of attachmentsare known in the art, particularly when a nucleotide comprisesderivatives or analogs of a naturally occurring 5-carbon sugar orphosphorus moiety.

D. Nucleic Acid Analogs

A nucleic acid may comprise, or be composed entirely of, a derivative oranalog of a nucleobase, a nucleobase linker moiety and/or backbonemoiety that may be present m a naturally occurring nucleic acid. RNAwith nucleic acid analogs may also be labeled according to methods ofthe invention As used herein a “derivative” refers to a chemicallymodified or altered form of a naturally occurring molecule, while theterms “mimic” or “analog” refer to a molecule that may or may notstructurally resemble a naturally occurring molecule or moiety, butpossesses similar functions. As used herein, a “moiety” generally refersto a smaller chemical or molecular component of a larger chemical ormolecular structure. Nucleobase, nucleoside and nucleotide analogs orderivatives are well known in the art, and have been described (see forexample, Scheit, 1980, incorporated herein by reference).

Additional non-limiting examples of nucleosides, nucleotides or nucleicacids comprising 5-carbon sugar and/or backbone moiety derivatives oranalogs, include those in U.S. Pat. No. 5,681,947, which describesoligonucleotides comprising purine derivatives that form triple helixeswith and/or prevent expression of dsDNA; U.S. Pat. Nos. 5,652,099 and5,763,167, which describe nucleic acids incorporating fluorescentanalogs of nucleosides found in DNA or RNA, particularly for use asfluorescent nucleic acids probes; U.S. Pat. No. 5,614,617, whichdescribes oligonucleotide analogs with substitutions on pyrimidine ringsthat possess enhanced nuclease stability, U.S. Pat. Nos. 5,670,663,5,872,232 and 5,859,221, which describe oligonucleotide analogs withmodified 5-carbon sugars (i.e., modified 2′-deoxyfuranosyl moieties)used in nucleic acid detection; U.S. Pat. No. 5,446,137, which describesoligonucleotides comprising at least one 5-carbon sugar moietysubstituted at the 4′ position with a substituent other than hydrogenthat can be used in hybridization assays, U.S. Pat. No. 5,886,165, whichdescribes oligonucleotides with both deoxyribonucleotides with 3′-5′mternucleotide linkages and ribonucleotides with 2′-5′ internucleotidelinkages, U.S. Pat. No. 5,714,606, which describes a modifiedinternucleotide linkage wherein a 3′-position oxygen of theinternucleotide linkage is replaced by a carbon to enhance the nucleaseresistance of nucleic acids; U.S. Pat. No. 5,672,697, which describesoligonucleotides containing one or more 5′ methylene phosphonatemternucleotide linkages that enhance nuclease resistance, U.S. Pat. Nos.5,466,786 and 5,792,847, which describe the linkage of a substituentmoiety which may comprise a drug or label to the 2′ carbon of anoligonucleotide to provide enhanced nuclease stability and ability todeliver drugs or detection moieties, U.S. Pat. No. 5,223,618, whichdescribes oligonucleotide analogs with a 2 or 3 carbon backbone linkageattaching the 4′ position and 3′ position of adjacent 5-carbon sugarmoiety to enhanced cellular uptake, resistance to nucleases andhybridization to target RNA, U.S. Pat. No. 5,470,967, which describesoligonucleotides composing at least one sulfamate or sulfamideinternucleotide linkage that are useful as nucleic acid hybridizationprobe; U.S. Pat. Nos. 5,378,825, 5,777,092, 5,623,070, 5,610,289 and5,602,240, which describe oligonucleotides with three or four atomlinker moiety replacing phosphodiester backbone moiety used for improvednuclease resistance, cellular uptake and regulating RNA expression, U.S.Pat. No. 5,858,988, which describes hydrophobic carrier agent attachedto the 2′-O position of oligonucleotides to enhanced their membranepermeability and stability; U.S. Pat. No. 5,214,136, which describesoligonucleotides conjugated to anthraqumone at the 5′ terminus thatpossess enhanced hybridization to DNA or RNA, enhanced stability tonucleases, U.S. Pat. No. 5,700,922, which describes PNA-DNA-PNA chimeraswherein the DNA comprises 2′-deoxy-erythro-pentofuranosyl nucleotidesfor enhanced nuclease resistance, binding affinity, and ability toactivate RNase H, and U.S. Pat. No. 5,708,154, which describes RNAlinked to a DNA to form a DNA-RNA hybrid, U.S. Pat. No. 5,728,525, whichdescribes the labeling of nucleoside analogs with a universalfluorescent label.

Additional teachings for nucleoside analogs and nucleic acid analogs areU.S. Pat. No. 5,728,525, which describes nucleoside analogs that areend-labeled, U.S. Pat. Nos. 5,637,683, 6,251,666 (L-nucleotidesubstitutions), and U.S. Pat. No. 5,480,980 (7-deaza-2′deoxyguanosinenucleotides and nucleic acid analogs thereof).

E. Modified Nucleotides

Labeling methods and kits of the invention specifically contemplate theuse of nucleotides that are both modified for attachment of a label andcan be incorporated into an miRNA molecule Such nucleotides includethose that can be labeled with a dye, including a fluorescent dye, orwith a molecule such as biotin. Labeled nucleotides are readilyavailable; they can be acquired commercially or they can be synthesizedby reactions known to those of skill in the art.

Modified nucleotides for use in the invention are not naturallyoccurring nucleotides, but instead, refer to prepared nucleotides thathave a reactive moiety on them. Specific reactive functionalities ofinterest include: ammo, sulfhydryl, sulfoxyl, ammosulfhydryl, azido,epoxide, isothiocyanate, isocyanate, anhydride, monochlorotitazme,dichlorotitazme, mono-or dihalogen substituted pyridine, mono- ordisubstituted diazme, maleimide, epoxide, aziπdme, sulfonyl halide, acidhahde, alkyl hahde, aryl hahde, alkylsulfonate, N-hydroxysuccimmideester, imido ester, hydrazine, azidomtrophenyl, azide, 3-(2-pyridyldithio)-propionarmde, glyoxal, aldehyde, lodoacetyl, cyanomethyl ester,p-nitrophenyl ester, o-mtrophenyl ester, hydroxypyridme ester, carbonylimidazole, and the other such chemical groups. In some embodiments, thereactive functionality may be bonded directly to a nucleotide, or it maybe bonded to the nucleotide through a linking group. The functionalmoiety and any linker cannot substantially impair the ability of thenucleotide to be added to the miRNA or to be labeled. Representativelinking groups include carbon containing linking groups, typicallyranging from about 2 to 18, usually from about 2 to 8 carbon atoms,where the carbon containing linking groups may or may not include one ormore heteroatoms, e.g. S, O, N etc., and may or may not include one ormore sites of unsaturation. Of particular interest in many embodimentsare alkyl linking groups, typically lower alkyl linking groups of 1 to16, usually 1 to 4 carbon atoms, where the linking groups may includeone or more sites of unsaturation. The functionalized nucleotides (orprimers) used in the above methods of functionalized target generationmay be fabricated using known protocols or purchased from commercialvendors, e.g., Sigma, Roche, Ambion, and NEN. Functional groups may beprepared according to ways known to those of skill in the art, includingthe representative information found in U.S. Pat Nos. 4,404,289;4,405,711; 4,337,063, 5,268,486 and Br. Pat. No. 1,529,202, which areall incorporated by reference.

Amine-modified nucleotides are used in several embodiments of theinvention. The amine-modified nucleotide is a nucleotide that has areactive amine group for attachment of the label. It is contemplatedthat any ribonucleotide (G, A, U, or C) or deoxyribonucleotide (G, A, T,or C) can be modified for labeling. Examples include, but are notlimited to, the following modified ribo- and deoxyribo-nucleotides:5-(3-aminoalryl)-UTP; 8-[(4-amino)butyl]-amino-ATP and8-[(6-amino)butyl]-amino-ATP; N6-(4-amino)butyl-ATP,N6-(6-amino)butyl-ATP, N4-[2,2-oxy-bis-(ethylamine)]-CTP;N6-(6-Amino)hexyl-ATP; 8-[(6-Amino)hexyl]-amino-ATP;5-propargylamino-CTP, 5-propargylamino-UTP; 5-(3-aminoallyl)-dUTP;8-[(4-amino)butyl]-amino-dATP and 8-[(6-amino)butyl]-amino-dATP;N6-(4-amino)butyl-dATP, N6-(6-amino)butyl-dATP, N6-(6-Amino)hexyl-dATP;8-[(6-Amino)hexyl]-amino-dATP; 5-propargylamino-dCTP, and5-propargylamino-dUTP. Such nucleotides can be prepared according tomethods known to those of skill in the art. Moreover, a person ofordinary skill in the art could prepare other nucleotide entities withthe same amine-modification, such as a 5-(3-aminoallyl)-CTP, GTP, ATP,dCTP, dGTP, dTTP, or dUTP in place of a 5-(3-aminoallyl)-UTP.

F. Preparation of Nucleic Acids

A nucleic acid may be made by any technique known to one of ordinaryskill in the art, such as for example, chemical synthesis, enzymaticproduction or biological production. In some embodiments, miRNAcompositions of the invention are chemically synthesized.

In some embodiments of the invention, miRNAs are recovered from abiological sample. The miRNA may be recombinant or it may be natural orendogenous to the cell (produced from the cell's genome). It iscontemplated that a biological sample may be treated in a way so as toenhance the recovery of small RNA molecules such as miRNA. U.S. patentapplication Ser. No. 10/667,126 describes such methods and it isspecifically incorporated by reference herein. Generally, methodsinvolve lysing cells with a solution having guanidinium and a detergent.

Alternatively, nucleic acid synthesis is performed according to standardmethods. See, for example, Itakura and Riggs (1980). Additionally, U.S.Pat. No. 4,704,362, U.S. Pat. No. 5,221,619, and U.S. Pat. No. 5,583,013each describe various methods of preparing synthetic nucleic acids.Non-limiting examples of a synthetic nucleic acid (e.g., a syntheticoligonucleotide), include a nucleic acid made by in vitro chemicallysynthesis using phosphotriester, phosphite or phosphoramidite chemistryand solid phase techniques such as described in EP 266,032, incorporatedherein by reference, or via deoxynucleoside H-phosphonate intermediatesas described by Froehler et al, 1986 and U.S. Pat. No. 5,705,629, eachincorporated herein by reference. In the methods of the presentinvention, one or more oligonucleotide may be used. Various differentmechanisms of oligonucleotide synthesis have been disclosed in forexample, U.S. Pat. Nos. 4,659,774, 4,816,571, 5,141,813, 5,264,566,4,959,463, 5,428,148, 5,554,744, 5,574,146, 5,602,244, each of which isincorporated herein by reference.

A non-limiting example of an enzymatically produced nucleic acid includeone produced by enzymes in amplification reactions such as PCR™ (see forexample, U.S. Pat. No. 4,683,202 and U.S. Pat. No. 4,682,195, eachincorporated herein by reference), or the synthesis of anoligonucleotide described in U.S. Pat. No. 5,645,897, incorporatedherein by reference. A non-limiting example of a biologically producednucleic acid includes a recombinant nucleic acid produced (i.e.,replicated) in a living cell, such as a recombinant DNA vectorreplicated in bacteria (see for example, Sambrook et al. 1989,incorporated herein by reference).

Oligonucleotide synthesis is well known to those of skill in the art.Various different mechanisms of oligonucleotide synthesis have beendisclosed in for example, U.S. Pat. Nos. 4,659,774, 4,816,571,5,141,813, 5,264,566, 4,959,463, 5,428,148, 5,554,744, 5,574,146,5,602,244, each of which is incorporated herein by reference.

Basically, chemical synthesis can be achieved by the diester method, thetriester method polynucleotides phosphorylase method and by solid-phasechemistry. These methods are discussed in further detail below.

Diester method. The diester method was the first to be developed to ausable state, primarily by Khorana and co-workers. (Khorana, 1979). Thebasic step is the joining of two suitably protected deoxynucleotides toform a dideoxynucleotide containing a phosphodiester bond. The diestermethod is well established and has been used to synthesize DNA molecules(Khorana, 1979).

Triester method. The main difference between the diester and triestermethods is the presence, in the latter of an extra protecting group onthe phosphate atoms of the reactants and products (Itakura et al.,1975). The phosphate protecting group is usually a chlorophenyl group,which renders the nucleotides and polynucleotide intermediates solublein organic solvents. Therefore purification's are done in chloroformsolutions. Other improvements in the method include (i) the blockcoupling of trimers and larger oligomers, (ii) the extensive use ofhigh-performance liquid chromatography for the purification of bothintermediate and final products, and (iii) solid-phase synthesis.

Polynucleotide phosphorylase method. This is an enzymatic method of DNAsynthesis that can be used to synthesize many useful oligonucleotides(Gillam et al., 1978; Gillam et al., 1979). Under controlled conditions,polynucleotide phosphorylase adds predominantly a single nucleotide to ashort oligonucleotide. Chromatographic purification allows the desiredsingle adduct to be obtained. At least a trimer is required to start theprocedure, and this primer must be obtained by some other method. Thepolynucleotide phosphorylase method works and has the advantage that theprocedures involved are familiar to most biochemists.

Solid-phase methods. Drawing on the technology developed for thesolid-phase synthesis of polypeptides, it has been possible to attachthe initial nucleotide to solid support material and proceed with thestepwise addition of nucleotides. All mixing and washing steps aresimplified, and the procedure becomes amenable to automation. Thesesyntheses are now routinely carried out using automatic nucleic acidsynthesizers.

Phosphoramidite chemistry (Beaucage and Lyer, 1992) has become by farthe most widely used coupling chemistry for the synthesis ofoligonucleotides. As is well known to those skilled in the art,phosphoramidite synthesis of oligonucleotides involves activation ofnucleoside phosphoramidite monomer precursors by reaction with anactivating agent to form activated intermediates, followed by sequentialaddition of the activated intermediates to the growing oligonucleotidechain (generally anchored at one end to a suitable solid support) toform the oligonucleotide product.

Recombinant methods. Recombinant methods for producing nucleic acids ina cell are well known to those of skill in the art. These include theuse of vectors (viral and non-viral), plasmids, cosmids, and othervehicles for delivering a nucleic acid to a cell, which may be thetarget cell or simply a host cell (to produce large quantities of thedesired RNA molecule). Alternatively, such vehicles can be used in thecontext of a cell free system so long as the reagents for generating theRNA molecule are present. Such methods include those described inSambrook, 2003, Sambrook, 2001 and Sambrook, 1989, which are herebyincorporated by reference.

In certain embodiments, the present invention concerns nucleic acidmolecules that are not synthetic. In some embodiments, the nucleic acidmolecule has a chemical structure of a naturally occurring nucleic acidand a sequence of a naturally occurring nucleic acid, such as the exactand entire sequence of a single stranded primary miRNA (see Lee 2002), asingle-stranded precursor miRNA, or a single-stranded mature miRNA. Inaddition to the use of recombinant technology, such non-syntheticnucleic acids may be generated chemically, such as by employingtechnology used for creating oligonucleotides.

G. Isolation of Nucleic Acids

Nucleic acids may be isolated using techniques well known to those ofskill in the art, though in particular embodiments, methods forisolating small nucleic acid molecules and/or isolating RNA moleculescan be employed. Chromatography is a process often used to separate orisolate nucleic acids from protein or from other nucleic acids. Suchmethods can involve electrophoresis with a gel matrix, filter columns,alcohol precipitation, and/or other chromatography. If miRNA from cellsis to be used or evaluated, methods generally involve lysing the cellswith a chaotropic (e.g., guanidinium isothiocyanate) and/or detergent(e.g., N-lauroyl sarcosine) prior to implementing processes forisolating particular populations of RNA.

In particular methods for separating miRNA from other nucleic acids, agel matrix is prepared using polyacrylamide, though agarose can also beused. The gels may be graded by concentration or they may be uniform.Plates or tubing can be used to hold the gel matrix for electrophoresis.Usually one-dimensional electrophoresis is employed for the separationof nucleic acids. Plates are used to prepare a slab gel, while thetubing (glass or rubber, typically) can be used to prepare a tube gel.The phrase “tube electrophoresis” refers to the use of a tube or tubing,instead of plates, to form the gel. Materials for implementing tubeelectrophoresis can be readily prepared by a person of skill in the artor purchased, such as from C.B.S. Scientific Co., Inc. or Scie-Plas.

Methods may involve the use of organic solvents and/or alcohol toisolate nucleic acids, particularly miRNA used in methods andcompositions of the invention. Some embodiments are described in U.S.patent application Ser. No. 10/667,126, which is hereby incorporated byreference.

In specific embodiments, miRNA isolation processes include: a) lysingcells in the sample with a lysing solution comprising guanidinium,wherein a lysate with a concentration of at least about 1 M guanidiniumis produced; b) extracting miRNA molecules from the lysate with anextraction solution comprising phenol; c) adding to the lysate analcohol solution for form a lysate/alcohol mixture, wherein theconcentration of alcohol in the mixture is between about 35% to about70%; d) applying the lysate/alcohol mixture to a solid support; e)eluting the miRNA molecules from the solid support with an ionicsolution; and, f) capturing the miRNA molecules. Typically the sample isdried down and resuspended in a liquid and volume appropriate forsubsequent manipulation.

V. Cancer Treatment

Cancers that may be treated or prevented by methods and compositions ofthe invention include cancer cells from the ovary, bladder, blood, bone,bone marrow, brain, breast, colon, esophagus, gastrointestine, gum,head, kidney, liver, lung, nasopharynx, neck, prostate, skin, stomach,testis, tongue, or uterus. In addition, the cancer may specifically beof the following histological type, though it is not limited to these:neoplasm, malignant; angiosarcomas; carcinoma; carcinoma,undifferentiated; giant and spindle cell carcinoma; small cellcarcinoma; papillary carcinoma; squamous cell carcinoma;lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma;transitional cell carcinoma; papillary transitional cell carcinoma;adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma;hepatocellular carcinoma; combined hepatocellular carcinoma andcholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma;adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposiscoli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolaradenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma;acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clearcell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma;papillary and follicular adenocarcinoma; nonencapsulating sclerosingcarcinoma; adrenal cortical carcinoma; endometroid carcinoma; skinappendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma;ceruminous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma;papillary cystadenocarcinoma; papillary serous cystadenocarcinoma;mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cellcarcinoma; infiltrating duct carcinoma; medullary carcinoma; lobularcarcinoma; inflammatory carcinoma; paget's disease, mammary; acinar cellcarcinoma; adenosquamous carcinoma; adenocarcinoma w/squamousmetaplasia; thymoma, malignant; ovarian stromal tumor, malignant;thecoma, malignant; granulosa cell tumor, malignant; androblastoma,malignant; Sertoli cell carcinoma; leydig cell tumor, malignant; lipidcell rumor, malignant; paraganglioma, malignant; extra-mammaryparaganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignantmelanoma; amelanotic melanoma; superficial spreading melanoma; maligmelanoma in giant pigmented nevus; epithelioid cell melanoma; bluenevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma,malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma;embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma;mixed tumor, malignant; mullerian mixed tumor; nephroblastoma;hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor,malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma,malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant;struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant;hemangiosarcoma; hemangioendothelioma, malignant; kaposi's sarcoma;hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma;juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant;mesenchymal chondrosarcoma; giant cell tumor of bone; ewing's sarcoma;odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma,malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma;glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma;fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma;oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma;ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactoryneurogenic tumor; meningioma, malignant; neurofibrosarcoma;neurilemmoma, malignant; granular cell tumor, malignant; malignantlymphoma; Hodgkin's disease; Hodgkin's lymphoma; paragranuloma;malignant lymphoma, small lymphocytic; malignant lymphoma, large cell,diffuse; malignant lymphoma, follicular; mycosis fungoides; otherspecified non-Hodgkin's lymphomas; malignant histiocytosis; multiplemyeloma; mast cell sarcoma; immunoproliferative small intestinaldisease; leukemia; lymphoid leukemia; plasma cell leukemia;erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia;basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mastcell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairycell leukemia. Moreover, miRNA can be utilized in a primary tumor or ametastasized tumor or to target specific subpopulations of cells withina cancer complex that may play a critical role in promoting itsresistance to therapy, progression, recurrence and/or metastasis. Thesecompartments or cells potentially include cancer stem cells, cellsinvolved in creating or inducing the creation of blood vessels,malignant epithelial cells, stromal or connective tissue cells importantfor and/or cells important for creating specific niches that promotecancer growth survival or metastasis.

In order to increase the effectiveness of a cancer treatment, it may bedesirable to combine the treatment with one or more miRNAs of theinvention effective to enhance the cancer treatment. A cancer treatmentis capable of negatively affecting cancer in a subject, for example, bykilling cancer cells, inducing apoptosis in cancer cells, reducing thegrowth rate of cancer cells, reducing the incidence or number ofmetastases, reducing tumor size, inhibiting tumor growth, reducing theblood supply to a tumor or cancer cells, promoting an immune responseagainst cancer cells or a tumor, preventing or inhibiting theprogression of cancer, or increasing the lifespan of a subject withcancer. More generally, these other compositions would be provided in acombined amount effective to kill or inhibit proliferation of the cell.In some embodiments, they could inhibit the ability of specific cells togenerate progeny capable of re-populating tumor recurrences, as cancerstem cells are now believed to do. Similarly, this may involve thedifferentiation of specific subpopulations of tumor cells into new typesof tissues, such as blood vessels capable of supporting continued tumorgrowth. These processes may involve contacting the cells with theexpression construct and the agent(s) or multiple factor(s) at the sametime. This may be achieved by contacting the cell with a singlecomposition or pharmacological formulation that includes both agents, orby contacting the cell with two distinct compositions or formulations,at the same time, wherein one composition includes the expressionconstruct and the other includes the second agent(s). In the context ofthe present invention, it is contemplated that microRNA therapy could beused similarly in conjunction with chemotherapeutic, immunotherapeutic,and/or hormone therapy intervention. It is expected that the treatmentcycles would be repeated as necessary. It also is contemplated thatvarious standard therapies, as well as surgical intervention, may beapplied in combination with the described hyperproliferative celltherapy. Exemplary additional therapies include surgery, chemotherapy,radiation, hormone therapy, immunotherapy, and or a combination thereof.

VI. Methods and Materials for Production of miRNA

The microRNAs of the invention can be isolated from cells or tissues,recombinantly produced, or synthesized in vitro by a variety oftechniques well known to one of ordinary skill in the art, for example.

In one embodiment, miRNA is isolated from cells or tissues. Techniquesfor isolating miRNA from cells or tissues are well known to one ofordinary skill in the art. For example, miRNA can be isolated from totalRNA using the mirVana miRNA isolation kit from Ambion, Inc. Anothertechnique utilizes the flashPAGE™ Fractionator System (Ambion, Inc.) forPAGE purification of small nucleic acids.

The miRNA can be obtained by preparing a recombinant version thereof(i.e., by using the techniques of genetic engineering to produce arecombinant nucleic acid which can then be isolated or purified bytechniques well known to one of ordinary skill in the art). Thisembodiment involves growing a culture of host cells in a suitableculture medium, and purifying the miRNA from the cells or the culture inwhich the cells are grown. For example, the methods include a processfor producing a miRNA in which a host cell containing a suitableexpression vector that includes a nucleic acid encoding a miRNA iscultured under conditions that allow expression of the encoded miRNA. Ina preferred embodiment the nucleic acid encodes the microRNA. The miRNAcan be recovered from the culture, from the culture medium or from alysate prepared from the host cells, and further purified. The host cellcan be a higher eukaryotic host cell such as a mammalian cell, a lowereukaryotic host cell such as a yeast cell, or the host cell can be aprokaryotic cell such as a bacterial cell. Introduction of a vectorcontaining the nucleic acid encoding the miRNA into the host cell can beeffected by calcium phosphate transfection, DEAE, dextran mediatedtransfection, or electroporation (Davis, L. et al., Basic Methods inMolecular Biology (1986)).

Any host/vector system can be used to express one or more of the miRNAs.These include, but are not limited to, eukaryotic hosts such as HeLacells and yeast, as well as prokaryotic host such as E. coli and B.subtilis. miRNA can be expressed in mammalian cells, yeast, bacteria, orother cells where the miRNA gene is under the control of an appropriatepromoter. Appropriate cloning and expression vectors for use withprokaryotic and eukaryotic hosts are described by Sambrook, et al., inMolecular Cloning: A Laboratory Manual, Second Edition, Cold SpringHarbor, N.Y. (1989). In the preferred embodiment, the miRNA is expressedin mammalian cells. Examples of mammalian expression systems includeC127, monkey COS cells, Chinese Hamster Ovary (CHO) cells, human kidney293 cells, human epidermal A431 cells, human Colo205 cells, 3T3 cells,CV-1 cells, other transformed primate cell lines, normal diploid cells,cell strains derived from in vitro culture of primary tissue, primaryexplants, HeLa cells, mouse L cells, BHK, HL-60, U937, HaK or Jurkatcells. Mammalian expression vectors may comprise an origin ofreplication, a suitable promoter, polyadenylation site, transcriptionaltermination sequences, and 5′ flanking nontranscribed sequences. DNAsequences derived from the SV40 viral genome, for example, SV40 origin,early promoter, enhancer, splice, and polyadenylation sites may be usedto provide the required nontranscribed genetic elements. Potentiallysuitable yeast strains include Saccharomyces cerevisiae,Schizosaccharomyces pombe, Kluyveromyces strains, Candida, or any yeaststrain capable of expressing miRNA. Potentially suitable bacterialstrains include Escherichia coli, Bacillus subtilis, Salmonellatyphimurium, or any bacterial strain capable of expressing miRNA.

The miRNA may be prepared by culturing transformed host cells underculture conditions suitable to express the miRNA. The resultingexpressed miRNA may then be purified from such culture (i.e., fromculture medium or cell extracts) using known purification processes,such as gel filtration and ion exchange chromatography. The purificationof the miRNA may also include an affinity column containing agents whichwill bind to the protein; one or more column steps over such affinityresins as concanavalin A-agarose, Heparin-Toyopearl™ or Cibacrom blue3GA Sepharose™; one or more steps involving hydrophobic interactionchromatography using such resins as phenyl ether, butyl ether, or propylether; immunoaffinity chromatography, or complementary cDNA affinitychromatography.

The miRNA may also be expressed as a product of transgenic animals,which are characterized by somatic or germ cells containing a nucleotidesequence encoding the miRNA. A vector containing DNA encoding miRNA andappropriate regulatory elements can be inserted in the germ line ofanimals using homologous recombination (Capecchi, Science 244:1288-1292(1989)), such that the express the miRNA. Transgenic animals, preferablynon-human mammals, are produced using methods as described in U.S. Pat.No. 5,489,743 to Robinson, et al., and PCT Publication No. WO 94/28122by Ontario Cancer Institute. miRNA can be isolated from cells or tissueisolated from transgenic animals as discussed above.

In a preferred embodiment, the miRNA can be obtained synthetically, forexample, by chemically synthesizing a nucleic acid by any method ofsynthesis known to the skilled artisan. The synthesized miRNA can thenbe purified by any method known in the art. Methods for chemicalsynthesis of nucleic acids include, but are not limited to, in vitrochemical synthesis using phosphotriester, phosphate or phosphoramiditechemistry and solid phase techniques, or via deosynucleosideH-phosphonate intermediates (see U.S. Pat. No. 5,705,629 to Bhongle).

In some circumstances, for example, where increased nuclease stabilityis desired, nucleic acids having nucleic acid analogs and/or modifiedinternucleoside linkages may be preferred. Nucleic acids containingmodified internucleoside linkages may also be synthesized using reagentsand methods that are well known in the art. For example, methods ofsynthesizing nucleic acids containing phosphonate phosphorothioate,phosphorodithioate, phosphoramidate methoxyethyl phosphoramidate,formacetal, thioformacetal, diisopropylsilyl, acetamidate, carbamate,dimethylene-sulfide (—CH₂—S—CH₂) dimethylene-sulfoxide (—CH₂—SO—CH₂),dimethylene-sulfone (—CH₂—S—CH₂), 2′-O-alkyl, and 2′-deoxy-2′-fluorophosphorothioate internucleoside linkages are well known in the art (seeUhlmann et al., 1990, Chem. Rev. 90:543-584; Schneider et al., 1990,Tetrahedron Lett. 31:335 and references cited therein). U.S. Pat. Nos.5,614,617 and 5,223,618 to Cook, et al., U.S. Pat. No. 5,714,606 toAcevedo, et al., U.S. Pat. No. 5,378,825 to Cook, et al., U.S. Pat. Nos.5,672,697 and 5,466,786 to Buhr, et al., U.S. Pat. No. 5,777,092 toCook, et al., U.S. Pat. No. 5,602,240 to De Mesmaeker, et al., U.S. Pat.No. 5,610,289 to Cook, et al. and U.S. Pat. No. 5,858,988 to Wang, alsodescribe nucleic acid analogs for enhanced nuclease stability andcellular uptake.

VII. Methods of Treatment

Methods for enhancement of treatment or prevention of at least onesymptom or manifestation of cancer are provided including administrationof an effective amount of a composition containing a microRNA miRNA520-a, g, and/or h nucleic acid molecule to alleviate at least onesymptom or decrease at least one manifestation. In an embodiment, thecancer is ovarian. The compositions described herein can be administeredin effective dosages alone or in combination with cancer therapy such aschemotherapy, immunotherapy, radiation therapy, and/or hormonal or othertargeted therapy to provide a beneficial effect for treatment, e.g.reduce the dosage of anti-cancer therapy and/or reduce the duration oftreatment and/or reduce the number of administrations of treatment;reduce cycle number, tumor size, reduce cell proliferation of the tumor,inhibit angiogenesis, inhibit metastasis, or otherwise improve at leastone symptom or manifestation of the disease. These can includecombinations of treatments that do not directly target tumor cells, butinstead enhance systemic responses designed to target tumor cells oralleviate symptoms, such as immunotherapy. Combinations of miR-520a/g/htreatments could be used to potentiate the response of interventionsdesigned to impact the immune response to a patient's cancer even thoughthe use of miR-520g/h/a treatment may or may not directly alter patternsof gene expression or responses of immune cells themselves. Mir-520g/h/ain combination with radiation therapy is used to enhance response totherapy, in particular aspects.

The compositions are administered to an individual in need of treatmentof at least one symptom or manifestation (since disease canoccur/progress in the absence of symptoms) of cancer. The individual maybe at risk for cancer, such as having personal or family history, be atobacco user, have genetic marker(s) and so forth. In some cases, thetherapy acts as a radiation or chemotherapy sensitizer, for example. Thecompositions described herein can be administered to a subject prior toadministration of a cytotoxic therapy in an amount effective tosensitize cells or tissues to be treated to the effects of the cytotoxictherapy. In one embodiment the cytotoxic therapy is radiotherapy. Inanother embodiment the cytotoxic therapy is chemotherapy. Sensitizationdescribes a condition of the cells or tissues to be treated in whichprior administration of the compositions described herein increases atleast one effect of the cytotoxic therapy on the cells or tissuesrelative to cells or tissues not receiving prior administration of thecompositions described herein. The increased effect may be on reductionof tumor size, reduction in cell proliferation of a tumor, inhibition ofangiogenesis, inhibition of metastasis, or improvement of at least onesymptom or manifestation of the disease.

VIII. Method of Administration

In general, methods of administering nucleic acids are well known in theart. In particular, the routes of administration already in use fornucleic acid therapeutics, along with formulations in current use,provide preferred routes of administration and formulation for thenucleic acids described above.

Nucleic acid compositions can be administered by a number of routesincluding, but not limited to: oral, intravenous, intraperitoneal,intramuscular, transdermal, subcutaneous, topical, sublingual, directtumor injection, or rectal means. Nucleic acids can also be administeredvia liposomes or nanoparticles. Such administration routes andappropriate formulations are generally known to those of skill in theart. In specific embodiments, the compositions of the invention areinjected intravenously as end modified miRNA mimics.

Administration of the formulations described herein may be accomplishedby any acceptable method that allows the miRNA or nucleic acid encodingthe miRNA to reach its target. The particular mode selected will dependof course, upon exemplary factors such as the particular formulation,the severity of the state of the subject being treated, and the dosagerequired for therapeutic efficacy. As generally used herein, an“effective amount” of a nucleic acid is the amount that is able to treatone or more symptoms of cancer or related disease, reverse theprogression of one or more symptoms of cancer or related disease, haltthe progression of one or more symptoms of cancer or related disease, orprevent the occurrence of one or more symptoms of cancer or relateddisease in a subject to whom the formulation is administered, ascompared to a matched subject not receiving the compound or therapeuticagent. The actual effective amounts of drug can vary according to thespecific drug or combination thereof being utilized, the particularcomposition formulated, the mode of administration, and the age, weight,condition of the patient, and severity of the symptoms or conditionbeing treated.

Any acceptable method known to one of ordinary skill in the art may beused to administer a formulation to the subject. The administration maybe localized (i.e., to a particular region, physiological system,tissue, organ, or cell type) or systemic, depending on the conditionbeing treated.

Injections can be e.g., intravenous, intradermal, subcutaneous,intramuscular, intratumoral, or intraperitoneal. The composition can beinjected intradermally for treatment or prevention of cancer, forexample. In some embodiments, the injections can be given at multiplelocations. Implantation includes inserting implantable drug deliverysystems, e.g., microspheres, hydrogels, polymeric reservoirs,cholesterol matrixes, polymeric systems, e.g., matrix erosion and/ordiffusion systems and non-polymeric systems, e.g., compressed, fused, orpartially-fused pellets. Inhalation includes administering thecomposition with an aerosol in an inhaler, either alone or attached to acarrier that can be absorbed. For systemic administration, it may bepreferred that the composition is encapsulated in liposomes.

Preferably, the agent and/or nucleic acid delivery system are providedin a manner which enables tissue-specific uptake of the agent and/ornucleic acid delivery system. Techniques include using tissue or organlocalizing devices, such as wound dressings or transdermal deliverysystems, using invasive devices such as vascular or urinary catheters,and using interventional devices such as stents having drug deliverycapability and configured as expansive devices or stent grafts.

The formulations may be delivered using a bioerodible implant by way ofdiffusion or by degradation of the polymeric matrix. In certainembodiments, the administration of the formulation may be designed so asto result in sequential exposures to the miRNA over a certain timeperiod, for example, hours, days, weeks, months or years. This may beaccomplished, for example, by repeated administrations of a formulationor by a sustained or controlled release delivery system in which themiRNA is delivered over a prolonged period without repeatedadministrations. Administration of the formulations using such adelivery system may be, for example, by oral dosage forms, bolusinjections, transdermal patches or subcutaneous implants. Maintaining asubstantially constant concentration of the composition may be preferredin some cases.

Other delivery systems suitable include, but are not limited to,time-release, delayed release, sustained release, or controlled releasedelivery systems. Such systems may avoid repeated administrations inmany cases, increasing convenience to the subject and the physician.Many types of release delivery systems are available and known to thoseof ordinary skill in the art. They include, for example, polymer-basedsystems such as polylactic and/or polyglycolic acids, polyanhydrides,polycaprolactones, copolyoxalates, polyesteramides, polyorthoesters,polyhydroxybutyric acid, and/or combinations of these. Microcapsules ofthe foregoing polymers containing nucleic acids are described in, forexample, U.S. Pat. No. 5,075,109. Other examples include nonpolymersystems that are lipid-based including sterols such as cholesterol,cholesterol esters, and fatty acids or neutral fats such as mono-, di-and triglycerides; hydrogel release systems; liposome-based systems;phospholipid based-systems; silastic systems; peptide based systems; waxcoatings; compressed tablets using conventional binders and excipients;or partially fused implants, or polymer-based compositions. Specificexamples include, but are not limited to, erosional systems in which themiRNA is contained in a formulation within a matrix (for example, asdescribed in U.S. Pat. Nos. 4,452,775, 4,675,189, 5,736,152, 4,667,013,4,748,034 and 5,239,660), or diffusional systems in which an activecomponent controls the release rate (for example, as described in U.S.Pat. Nos. 3,832,253, 3,854,480, 5,133,974 and 5,407,686). Theformulation may be as, for example, microspheres, hydrogels, polymericreservoirs, cholesterol matrices, or polymeric systems. In someembodiments, the system may allow sustained or controlled release of thecomposition to occur, for example, through control of the diffusion orerosion/degradation rate of the formulation containing the miRNA. Inaddition, a pump-based hardware delivery system may be used to deliverone or more embodiments.

Examples of systems in which release occurs in bursts includes, e.g.,systems in which the composition is entrapped in liposomes which areencapsulated in a polymer matrix, the liposomes being sensitive tospecific stimuli, e.g., temperature, pH, light or a degrading enzyme andsystems in which the composition is encapsulated by an ionically-coatedmicrocapsule with a microcapsule core degrading enzyme. Examples ofsystems in which release of the inhibitor is gradual and continuousinclude, e.g., erosional systems in which the composition is containedin a form within a matrix and effusional systems in which thecomposition permeates at a controlled rate, e.g., through a polymer.Such sustained release systems can be e.g., in the form of pellets, orcapsules.

Use of a long-term release implant may be particularly suitable in someembodiments. “Long-term release,” as used herein, means that the implantcontaining the composition is constructed and arranged to delivertherapeutically effective levels of the composition for at least 30 or45 days, and preferably at least 60 or 90 days, or even longer in somecases. Long-term release implants are well known to those of ordinaryskill in the art, and include some of the release systems describedabove.

Dosages for a particular patient can be determined by one of ordinaryskill in the art using conventional considerations, (e.g. by means of anappropriate, conventional pharmacological protocol). A physician may,for example, prescribe a relatively low dose at first, subsequentlyincreasing the dose until an appropriate response is obtained. Thedose-administered to a patient is sufficient to effect a beneficialtherapeutic response in the patient over time, or, e.g., to reducesymptoms, or other appropriate activity, depending on the application.The dose is determined by the efficacy of the particular formulation,and the activity, stability or serum half-life of the miRNA employed andthe condition of the patient, as well as the body weight or surface areaof the patient to be treated. The size of the dose is also determined bythe existence, nature, and extent of any adverse side-effects thataccompany the administration of a particular vector, and formulation, ina particular patient.

Therapeutic compositions comprising one or more nucleic acids areoptionally tested in one or more appropriate in vitro and/or in vivoanimal models of disease, to confirm efficacy, tissue metabolism, and toestimate dosages, according to methods well known in the art. Inparticular, dosages can be initially determined by activity, stabilityor other suitable measures of treatment versus non-treatment (e.g.,comparison of treated vs. untreated cells or animal models), in arelevant assay. Formulations are administered at a rate determined bythe LD50 of the relevant formulation, and/or observation of anyside-effects of the nucleic acids at various concentrations, e.g., asapplied to the mass and overall health of the patient. Administrationcan be accomplished via single or divided doses.

In vitro models can be used to determine the effective doses of thenucleic acids as a potential cancer treatment. Suitable in vitro modelsinclude, but are not limited to, proliferation assays of cultured tumorcells, growth of cultured tumor cells in soft agar (see Freshney, (1987)Culture of Animal Cells: A Manual of Basic Technique, Wily-Liss, NewYork, N.Y. Ch 18 and Ch 21), tumor systems in nude mice as described inGiovanella et al., J. Natl. Can. Inst., 52: 921-30 (1974), mobility andinvasive potential of tumor cells in Boyden Chamber assays as describedin Pilkington et al., Anticancer Res., 17: 4107-9 (1997), andangiogenesis assays such as induction of vascularization of the chickchorioallantoic membrane or induction of vascular endothelial cellmigration as described in Ribatta et al., Intl. JS. Dev. Biol., 40:1189-97 (1999) and Li et al., Clin. Exp. Metastasis, 17:423-9 (1999),respectively. Suitable tumor cells lines are available, e.g. fromAmerican Type Tissue Culture Collection catalogs.

In vivo models are the preferred models to determine the effective dosesof nucleic acids described above as potential cancer treatments.Suitable in vivo models include, but are not limited to, mice that carrya mutation in the KRAS oncogene (Lox-Stop-Lox K-Ras^(G12D) mutants,Kras2^(tm4Tyj)) available from the National Cancer Institute (NCI)Frederick Mouse Repository. Other mouse models known in the art and thatare available include but are not limited to models for gastrointestinalcancer, hematopoietic cancer, lung cancer, mammary gland cancer, nervoussystem cancer, ovarian cancer, prostate cancer, skin cancer, cervicalcancer, oral cancer, and sarcoma cancer.

In determining the effective amount of the miRNA to be administered inthe treatment or prophylaxis of disease the physician evaluatescirculating plasma levels, formulation toxicities, and progression ofthe disease.

The dose administered to a 70 kilogram patient is typically in the rangeequivalent to dosages of currently-used therapeutic antisenseoligonucleotides such as Vitravene® (fomivirsen sodium injection) whichis approved by the FDA for treatment of cytomegaloviral RNA, adjustedfor the altered activity or serum half-life of the relevant composition.

The formulations described herein can supplement treatment conditions byany known conventional therapy, including, but not limited to, antibodyadministration, vaccine administration, administration of cytotoxicagents, natural amino acid polypeptides, nucleic acids, nucleotideanalogues, and biologic response modifiers. Two or more combinedcompounds may be used together or sequentially. For example, the nucleicacids can also be administered in therapeutically effective amounts as aportion of an anti-cancer cocktail. An anti-cancer cocktail is a mixtureof the oligonucleotide or modulator with one or more anti-cancer drugsin addition to a pharmaceutically acceptable carrier for delivery. Theuse of anti-cancer cocktails as a cancer treatment is routine.Anti-cancer drugs that are well known in the art and can be used as atreatment in combination with the nucleic acids described hereininclude, but are not limited to: Actinomycin D, Aminoglutethimide,Asparaginase, Bleomycin, Busulfan, Carboplatin, Carmustine,Chlorambucil, Cisplatin (cis-DDP), Cyclophosphamide, Cytarabine HCl(Cytosine arabinoside), Dacarbazine, Dactinomycin, Daunorubicin HCl,Doxorubicin HCl, Estramustine phosphate sodium, Etoposide (V16-213),Floxuridine, 5-Fluorouracil (5-Fu), Flutamide, Hydroxyurea(hydroxycarbamide), Ifosfamide, Interferon Alpha-2a, InterferonAlpha-2b, Leuprolide acetate (LHRH-releasing factor analog), Lomustine,Mechlorethamine HC1 (nitrogen mustard), Melphalan, Mercaptopurine,Mesna, Methotrexate (MTX), Mitomycin, Mitoxantrone HCl, Octreotide,Plicamycin, Procarbazine HCl, Streptozocin, Tamoxifen citrate,Thioguanine, Thiotepa, Vinblastine sulfate, Vincristine sulfate,Amsacrine, Azacitidine, Hexamethylmelamine, Interleukin-2, Mitoguazone,Pentostatin, Semustine, Teniposide, and Vindesine sulfate.

IX. Chemotherapeutic Agents

These can be, for example, agents that directly cross-link DNA, agentsthat intercalate into DNA, and agents that lead to chromosomal andmitotic aberrations by affecting nucleic acid synthesis. Agents thatdirectly cross-link nucleic acids, specifically DNA, are envisaged andare shown herein, to eventuate DNA damage leading to a synergisticantineoplastic combination. Agents such as cisplatin, and other DNAalkylating agents may be used. Agents that damage DNA also includecompounds that interfere with DNA replication, mitosis, and chromosomalsegregation. Examples of these compounds include adriamycin (also knownas doxorubicin), VP-16 (also known as etoposide), podophyllotoxin, andthe like. Widely used in clinical setting for the treatment ofneoplasms, these compounds are administered, for example, through bolusinjections intravenously at doses ranging from 25-100 mg/m² at 21 dayintervals for adriamycin, to 35-100 mg/m² for etoposide intravenously ororally.

Examples of alternate strategies for targeting ovarian cancer includeagents that inhibit the activity of poly ADP ribose polymerase (PARP),such as Iniparib (BSI 201), Olaparib (AZD-2281) Rucaparib (AG014699,PF-01367338), Veliparib, MK4827 or BMN673. These agents inhibit theactivity of the gene product PARP1 that is involved in the repair ofsingle-strand DNA breaks. If single stranded DNA nicks persistunrepaired, events involved in DNA replication will cause double strandbreaks to form and ultimately result in cell death as injured cellsattempt to proceed through mitosis. In particular, cells with defects inhomologous recombination, including cells defective in BRCA1/2, Rad51and others are particularly sensitive to the biologic effects of PARPinhibitors, as cells with these defects are unable repair the doublestranded DNA breaks that ultimately result from inhibition of PARP1activity. In contrast, healthy cells able to reapir double stranded DNAnicks are relatively insensitive to these agents, hence the descriptionof this strategy in the art as “synthetic lethal”. In certainembodiments of the invention, one or more synthetic lethal compositionsare employed in conjunction with the miRNAs of the invention, either insuccession or at the same time.

Exemplary chemotherapeutics include at least 1) antibiotics, such asdoxorubicin, daunorubicin, mitomycin, Actinomycin D; 2) platinum-basedagents, such as cisplatin; 3) plant alkaloids, such as taxol andvincristine, vinblastine; 4) alkylating agents, such as carmustine,melphalin, cyclophosphamide, chlorambucil, bisufan, an dlomustine.

X. Immunotherapy

In some embodiments of the invention, one or more microRNAs are employedto enhance immunotherapy cancer treatment in an individual. In specificaspects, the immunotherapy comprises monoclonal antibodies, immunemodulating agents known to modify sensitivity or function of the immuneresponse, administration of cytokines or cytokine derivatives known toother agents or mileus in which immune responses to cancers and otherdiseases are generated, administration of adjuvants designed to enhancethe immunogenticity of specific antigents to challenge or modify immuneresponses.

Several of the newer chemotherapy agents are monoclonal antibodies.Monoclonal antibodies work by attaching to certain parts of thetumor-specific antigens and make them easily recognizable to the body'simmune system. Some prevent growth of cancer tumors by blocking the cellreceptors to the body's “growth factors”.

The first one was approved for cancer treatment by the Food and DrugAdministration (FDA) in 1997. Alemtuzumab (Campath®), Bevacizumab(Avastin®), Cetuximab (Erbitux®), Gemtuzumab (Mylotarg®), Ibritumomab(Zevalin®), Panitumumab (Vectibix®), Rituximab (Rituxan®), Tositumomab(Bexxar®), and Trastuzumab (Herceptin®) are some of the FDA-approvedmonoclonal drugs used in cancer treatments.

Monoclonal antibodies (abbreviated MAbs) are useful in treating at leastcolon, lung, head, neck, and breast cancers. Monoclonal drugs are alsoused to treat chronic lymphocytic leukemia, acute myelogenous leukemia,and non-Hodgkin's lymphoma.

XI. Hormone Therapy

In some embodiments of the invention, one or more microRNAs are employedto enhance hormone therapy cancer treatment in an individual. Hormonetherapy is a form of systemic therapy that is most often used to helpreduce the risk of the cancer coming back after surgery, but it may alsobe used for cancer that has spread or come back after treatment, forexample. The therapy may include drugs to block hormones or drugs thatchange hormone levels.

The female hormone estrogen, for example promotes the growth of breastcancer cells in some women (those who have hormone receptor-positivecancers). For these women, actions are taken to block the effect ofestrogen or lower its levels in order to treat breast cancer. Drugs usedto block estrogen Tamoxifen and toremifene (Fareston®): Drugs liketamoxifen can be given to block estrogen. Tamoxifen is taken in pill orliquid form, usually every day for up to 5 years after surgery, toreduce the risk the cancer will come back. This drug helps women withearly breast cancer if their cancer has hormone receptors (isER-positive or PR-positive). It is also used to treat hormonereceptor-positive breast cancer that has spread and to reduce the riskof breast cancer in women who are at high risk.

Toremifene works like tamoxifen, but is not used as often and is onlyapproved for patients with metastatic breast cancer. The side effects ofthese drugs are similar.

Fulvestrant: Fulvestrant (Faslodex®) is a drug that blocks the estrogenreceptor and then damages it. It often works even if the breast canceris no longer responding to tamoxifen. It is given as a shot once amonth. Hot flashes, mild nausea, and tiredness are the major sideeffects. Right now it is only used in postmenopausal women with advancedbreast cancer that no longer responds to tamoxifen or toremifene.

Drugs used to change hormone that include both direct receptorantagonists (tamoxifen, flutamide), selective receptoragonists/antagonists, agents that target steroid receptor co-activatorsor co-repressors as well as other agents designed to impact or altercirculating levels of hormones including levels include aromataseinhibitors (AIs), such as: anastrazole (Arimidex®), exemestane(Aromasin®), and letrozole (Femara®), for example. Androgens andandrogen antagonists may be employed.

XII. Pharmaceutical Preparations

Pharmaceutical compositions of the present invention comprise aneffective amount of one or more microRNAs dissolved or dispersed in apharmaceutically acceptable carrier. The phrases “pharmaceutical orpharmacologically acceptable” refers to molecular entities andcompositions that do not produce an adverse, allergic or other untowardreaction when administered to an animal, such as, for example, a human,as appropriate. The preparation of an pharmaceutical composition thatcontains at least one microRNA or additional active ingredient will beknown to those of skill in the art in light of the present disclosure,as exemplified by Remington's Pharmaceutical Sciences, 18th Ed. MackPrinting Company, 1990, incorporated herein by reference. Moreover, foranimal (e.g., human) administration, it will be understood thatpreparations should meet sterility, pyrogenicity, general safety andpurity standards as required by FDA Office of Biological Standards.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, surfactants, antioxidants,preservatives (e.g., antibacterial agents, antifungal agents), isotonicagents, absorption delaying agents, salts, preservatives, drugs, drugstabilizers, gels, binders, excipients, disintegration agents,lubricants, sweetening agents, flavoring agents, dyes, such likematerials and combinations thereof, as would be known to one of ordinaryskill in the art (see, for example, Remington's Pharmaceutical Sciences,18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated hereinby reference). Except insofar as any conventional carrier isincompatible with the active ingredient, its use in the pharmaceuticalcompositions is contemplated.

The microRNA may comprise different types of carriers depending onwhether it is to be administered in solid, liquid or aerosol form, andwhether it need to be sterile for such routes of administration asinjection. The present invention can be administered intravenously,intradermally, transdermally, intrathecally, intraarterially,intraperitoneally, intranasally, intravaginally, intrarectally,topically, intramuscularly, subcutaneously, mucosally, orally,topically, locally, inhalation (e.g., aerosol inhalation), injection,infusion, continuous infusion, localized perfusion bathing target cellsdirectly, via a catheter, via a lavage, in cremes, in lipid compositions(e.g., liposomes), or by other method or any combination of the forgoingas would be known to one of ordinary skill in the art (see, for example,Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company,1990, incorporated herein by reference).

The microRNA may be formulated into a composition in a free base,neutral or salt form. Pharmaceutically acceptable salts, include theacid addition salts, e.g., those formed with the free amino groups of aproteinaceous composition, or which are formed with inorganic acids suchas for example, hydrochloric or phosphoric acids, or such organic acidsas acetic, oxalic, tartaric or mandelic acid. Salts formed with the freecarboxyl groups can also be derived from inorganic bases such as forexample, sodium, potassium, ammonium, calcium or ferric hydroxides; orsuch organic bases as isopropylamine, trimethylamine, histidine orprocaine. Upon formulation, solutions will be administered in a mannercompatible with the dosage formulation and in such amount as istherapeutically effective. The formulations are easily administered in avariety of dosage forms such as formulated for parenteraladministrations such as injectable solutions, or aerosols for deliveryto the lungs, or formulated for alimentary administrations such as drugrelease capsules and the like.

Further in accordance with the present invention, the composition of thepresent invention suitable for administration is provided in apharmaceutically acceptable carrier with or without an inert diluent.The carrier should be assimilable and includes liquid, semi-solid, i.e.,pastes, or solid carriers. Except insofar as any conventional media,agent, diluent or carrier is detrimental to the recipient or to thetherapeutic effectiveness of a composition contained therein, its use inadministrable composition for use in practicing the methods of thepresent invention is appropriate. Examples of carriers or diluentsinclude fats, oils, water, saline solutions, lipids, liposomes, resins,binders, fillers and the like, or combinations thereof. The compositionmay also comprise various antioxidants to retard oxidation of one ormore component. Additionally, the prevention of the action ofmicroorganisms can be brought about by preservatives such as variousantibacterial and antifungal agents, including but not limited toparabens (e.g., methylparabens, propylparabens), chlorobutanol, phenol,sorbic acid, thimerosal or combinations thereof.

In accordance with the present invention, the composition is combinedwith the carrier in any convenient and practical manner, i.e., bysolution, suspension, emulsification, admixture, encapsulation,absorption and the like. Such procedures are routine for those skilledin the art.

In a specific embodiment of the present invention, the composition iscombined or mixed thoroughly with a semi-solid or solid carrier. Themixing can be carried out in any convenient manner such as grinding.Stabilizing agents can be also added in the mixing process in order toprotect the composition from loss of therapeutic activity, i.e.,denaturation in the stomach. Examples of stabilizers for use in an thecomposition include buffers, amino acids such as glycine and lysine,carbohydrates such as dextrose, mannose, galactose, fructose, lactose,sucrose, maltose, sorbitol, mannitol, etc.

In further embodiments, the present invention may concern the use of apharmaceutical lipid vehicle compositions that include microRNA, one ormore lipids, and an aqueous solvent. These can also be combined intomulti-stage vectors that have both liposomes as well as carriers forthose liposomes that may be of any range of a material including siliconand others. As used herein, the term “lipid” will be defined to includeany of a broad range of substances that is characteristically insolublein water and extractable with an organic solvent. This broad class ofcompounds are well known to those of skill in the art, and as the term“lipid” is used herein, it is not limited to any particular structure.Examples include compounds which contain long-chain aliphatichydrocarbons and their derivatives. A lipid may be naturally occurringor synthetic (i.e., designed or produced by man). However, a lipid isusually a biological substance. Biological lipids are well known in theart, and include for example, neutral fats, phospholipids,phosphoglycerides, steroids, terpenes, lysolipids, glycosphingolipids,glycolipids, sulphatides, lipids with ether and ester-linked fatty acidsand polymerizable lipids, and combinations thereof. Of course, compoundsother than those specifically described herein that are understood byone of skill in the art as lipids are also encompassed by thecompositions and methods of the present invention.

One of ordinary skill in the art would be familiar with the range oftechniques that can be employed for dispersing a composition in a lipidvehicle. For example, the microRNA may be dispersed in a solutioncontaining a lipid, dissolved with a lipid, emulsified with a lipid,mixed with a lipid, combined with a lipid, covalently bonded to a lipid,contained as a suspension in a lipid, contained or complexed with amicelle or liposome, or otherwise associated with a lipid or lipidstructure by any means known to those of ordinary skill in the art. Thedispersion may or may not result in the formation of liposomes.

The actual dosage amount of a composition of the present inventionadministered to an animal patient can be determined by physical andphysiological factors such as body weight, severity of condition, thetype of disease being treated, previous or concurrent therapeuticinterventions, idiopathy of the patient and on the route ofadministration. Depending upon the dosage and the route ofadministration, the number of administrations of a preferred dosageand/or an effective amount may vary according to the response of thesubject. The practitioner responsible for administration will, in anyevent, determine the concentration of active ingredient(s) in acomposition and appropriate dose(s) for the individual subject.

In certain embodiments, pharmaceutical compositions may comprise, forexample, at least about 0.1% of an active compound. In otherembodiments, the an active compound may comprise between about 2% toabout 75% of the weight of the unit, or between about 25% to about 60%,for example, and any range derivable therein. Naturally, the amount ofactive compound(s) in each therapeutically useful composition may beprepared is such a way that a suitable dosage will be obtained in anygiven unit dose of the compound. Factors such as solubility,bioavailability, biological half-life, route of administration, productshelf life, as well as other pharmacological considerations will becontemplated by one skilled in the art of preparing such pharmaceuticalformulations, and as such, a variety of dosages and treatment regimensmay be desirable.

In other non-limiting examples, a dose may also comprise from about 1microgram/kg/body weight, about 5 microgram/kg/body weight, about 10microgram/kg/body weight, about 50 microgram/kg/body weight, about 100microgram/kg/body weight, about 200 microgram/kg/body weight, about 350microgram/kg/body weight, about 500 microgram/kg/body weight, about 1milligram/kg/body weight, about 5 milligram/kg/body weight, about 10milligram/kg/body weight, about 50 milligram/kg/body weight, about 100milligram/kg/body weight, about 200 milligram/kg/body weight, about 350milligram/kg/body weight, about 500 milligram/kg/body weight, to about1000 mg/kg/body weight or more per administration, and any rangederivable therein. In non-limiting examples of a derivable range fromthe numbers listed herein, a range of about 5 mg/kg/body weight to about100 mg/kg/body weight, about 5 microgram/kg/body weight to about 500milligram/kg/body weight, etc., can be administered, based on thenumbers described above.

A. Alimentary Compositions and Formulations

In preferred embodiments of the present invention, the microRNA areformulated to be administered via an alimentary route. Alimentary routesinclude all possible routes of administration in which the compositionis in direct contact with the alimentary tract. Specifically, thepharmaceutical compositions disclosed herein may be administered orally,buccally, rectally, or sublingually. As such, these compositions may beformulated with an inert diluent or with an assimilable edible carrier,or they may be enclosed in hard- or soft-shell gelatin capsule, or theymay be compressed into tablets, or they may be incorporated directlywith the food of the diet.

In certain embodiments, the active compounds may be incorporated withexcipients and used in the form of ingestible tablets, buccal tables,troches, capsules, elixirs, suspensions, syrups, wafers, and the like(Mathiowitz et al., 1997; Hwang et al., 1998; U.S. Pat. Nos. 5,641,515;5,580,579 and 5,792, 451, each specifically incorporated herein byreference in its entirety). The tablets, troches, pills, capsules andthe like may also contain the following: a binder, such as, for example,gum tragacanth, acacia, cornstarch, gelatin or combinations thereof; anexcipient, such as, for example, dicalcium phosphate, mannitol, lactose,starch, magnesium stearate, sodium saccharine, cellulose, magnesiumcarbonate or combinations thereof; a disintegrating agent, such as, forexample, corn starch, potato starch, alginic acid or combinationsthereof; a lubricant, such as, for example, magnesium stearate; asweetening agent, such as, for example, sucrose, lactose, saccharin orcombinations thereof; a flavoring agent, such as, for examplepeppermint, oil of wintergreen, cherry flavoring, orange flavoring, etc.When the dosage unit form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier. Various other materialsmay be present as coatings or to otherwise modify the physical form ofthe dosage unit. For instance, tablets, pills, or capsules may be coatedwith shellac, sugar, or both. When the dosage form is a capsule, it maycontain, in addition to materials of the above type, carriers such as aliquid carrier. Gelatin capsules, tablets, or pills may be entericallycoated. Enteric coatings prevent denaturation of the composition in thestomach or upper bowel where the pH is acidic. See, e.g., U.S. Pat. No.5,629,001. Upon reaching the small intestines, the basic pH thereindissolves the coating and permits the composition to be released andabsorbed by specialized cells, e.g., epithelial enterocytes and Peyer'spatch M cells. A syrup of elixir may contain the active compound sucroseas a sweetening agent methyl and propylparabens as preservatives, a dyeand flavoring, such as cherry or orange flavor. Of course, any materialused in preparing any dosage unit form should be pharmaceutically pureand substantially non-toxic in the amounts employed. In addition, theactive compounds may be incorporated into sustained-release preparationand formulations.

For oral administration the compositions of the present invention mayalternatively be incorporated with one or more excipients in the form ofa mouthwash, dentifrice, buccal tablet, oral spray, or sublingualorally-administered formulation. For example, a mouthwash may beprepared incorporating the active ingredient in the required amount inan appropriate solvent, such as a sodium borate solution (Dobell'sSolution). Alternatively, the active ingredient may be incorporated intoan oral solution such as one containing sodium borate, glycerin andpotassium bicarbonate, or dispersed in a dentifrice, or added in atherapeutically-effective amount to a composition that may includewater, binders, abrasives, flavoring agents, foaming agents, andhumectants. Alternatively the compositions may be fashioned into atablet or solution form that may be placed under the tongue or otherwisedissolved in the mouth.

Additional formulations which are suitable for other modes of alimentaryadministration include suppositories. Suppositories are solid dosageforms of various weights and shapes, usually medicated, for insertioninto the rectum. After insertion, suppositories soften, melt or dissolvein the cavity fluids. In general, for suppositories, traditionalcarriers may include, for example, polyalkylene glycols, triglyceridesor combinations thereof. In certain embodiments, suppositories may beformed from mixtures containing, for example, the active ingredient inthe range of about 0.5% to about 10%, and preferably about 1% to about2%.

B. Parenteral Compositions and Formulations

In further embodiments, microRNA may be administered via a parenteralroute. As used herein, the term “parenteral” includes routes that bypassthe alimentary tract. Specifically, the pharmaceutical compositionsdisclosed herein may be administered for example, but not limited tointravenously, intradermally, intramuscularly, intraarterially,intrathecally, subcutaneous, or intraperitoneally U.S. Pat. Nos.6,7537,514, 6,613,308, 5,466,468, 5,543,158; 5,641,515; and 5,399,363(each specifically incorporated herein by reference in its entirety).

Solutions of the active compounds as free base or pharmacologicallyacceptable salts may be prepared in water suitably mixed with asurfactant, such as hydroxypropylcellulose. Dispersions may also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms. The pharmaceutical forms suitable for injectable useinclude sterile aqueous solutions or dispersions and sterile powders forthe extemporaneous preparation of sterile injectable solutions ordispersions (U.S. Pat. No. 5,466,468, specifically incorporated hereinby reference in its entirety). In all cases the form must be sterile andmust be fluid to the extent that easy injectability exists. It must bestable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms, such asbacteria and fungi. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (i.e., glycerol,propylene glycol, and liquid polyethylene glycol, and the like),suitable mixtures thereof, and/or vegetable oils. Proper fluidity may bemaintained, for example, by the use of a coating, such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. The prevention of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars or sodium chloride.Prolonged absorption of the injectable compositions can be brought aboutby the use in the compositions of agents delaying absorption, forexample, aluminum monostearate and gelatin.

For parenteral administration in an aqueous solution, for example, thesolution should be suitably buffered if necessary and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous, and intraperitoneal administration. In thisconnection, sterile aqueous media that can be employed will be known tothose of skill in the art in light of the present disclosure. Forexample, one dosage may be dissolved in isotonic NaCl solution andeither added hypodermoclysis fluid or injected at the proposed site ofinfusion, (see for example, “Remington's Pharmaceutical Sciences” 15thEdition, pages 1035-1038 and 1570-1580). Some variation in dosage willnecessarily occur depending on the condition of the subject beingtreated. The person responsible for administration will, in any event,determine the appropriate dose for the individual subject. Moreover, forhuman administration, preparations should meet sterility, pyrogenicity,general safety and purity standards as required by FDA Office ofBiologics standards.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof. A powdered composition is combined with a liquidcarrier such as, e.g., water or a saline solution, with or without astabilizing agent.

C. Miscellaneous Pharmaceutical Compositions and Formulations

In other preferred embodiments of the invention, the active compoundmicroRNA may be formulated for administration via various miscellaneousroutes, for example, topical (i.e., transdermal) administration, mucosaladministration (intranasal, vaginal, etc.) and/or inhalation.

Pharmaceutical compositions for topical administration may include theactive compound formulated for a medicated application such as anointment, paste, cream or powder. Ointments include all oleaginous,adsorption, emulsion and water-solubly based compositions for topicalapplication, while creams and lotions are those compositions thatinclude an emulsion base only. Topically administered medications maycontain a penetration enhancer to facilitate adsorption of the activeingredients through the skin. Suitable penetration enhancers includeglycerin, alcohols, alkyl methyl sulfoxides, pyrrolidones andluarocapram. Possible bases for compositions for topical applicationinclude polyethylene glycol, lanolin, cold cream and petrolatum as wellas any other suitable absorption, emulsion or water-soluble ointmentbase. Topical preparations may also include emulsifiers, gelling agents,and antimicrobial preservatives as necessary to preserve the activeingredient and provide for a homogenous mixture. Transdermaladministration of the present invention may also comprise the use of a“patch”. For example, the patch may supply one or more active substancesat a predetermined rate and in a continuous manner over a fixed periodof time.

In certain embodiments, the pharmaceutical compositions may be deliveredby eye drops, intranasal sprays, inhalation, and/or other aerosoldelivery vehicles. Methods for delivering compositions directly to thelungs via nasal aerosol sprays has been described e.g., in U.S. Pat.Nos. 5,756,353 and 5,804,212 (each specifically incorporated herein byreference in its entirety). Likewise, the delivery of drugs usingintranasal microparticle resins (Takenaga et al., 1998) andlysophosphatidyl-glycerol compounds (U.S. Pat. No. 5,725, 871,specifically incorporated herein by reference in its entirety) are alsowell-known in the pharmaceutical arts. Likewise, transmucosal drugdelivery in the form of a polytetrafluoroetheylene support matrix isdescribed in U.S. Pat. No. 5,780,045 (specifically incorporated hereinby reference in its entirety).

The term aerosol refers to a colloidal system of finely divided solid ofliquid particles dispersed in a liquefied or pressurized gas propellant.The typical aerosol of the present invention for inhalation will consistof a suspension of active ingredients in liquid propellant or a mixtureof liquid propellant and a suitable solvent. Suitable propellantsinclude hydrocarbons and hydrocarbon ethers. Suitable containers willvary according to the pressure requirements of the propellant.Administration of the aerosol will vary according to subject's age,weight and the severity and response of the symptoms.

XIII. Kits of the Invention

Kits are also included as part of the invention. Kits for implementingmethods of the invention described herein are specifically contemplated.In some embodiments, there are kits for treating and/or preventingcancer. In some embodiments, the kit comprises one or more 19q13.41microRNAs and optionally a cancer treatment.

In some embodiments, kit comprises in suitable container means, one ormore of the following: 1) poly(A) polymerase; 2) nucleotides (G, A, T,C, and/or U); 3) poly(A) polymerase buffer; reaction buffer; 4)solutions for preparing, isolating, enriching, and/or purifying miRNAs.Other reagents include those generally used for manipulating RNA, suchas formamide, loading dye, ribonuclease inhibitors, and DNase. Buffers,as well as other solutions, are contemplated to have a pH of about, atleast about, or at most about 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7,5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1,7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5,8.6, 8.7, 8.8, 8.9, 9.0 or more (or any range derivable therein) incertain embodiments of the invention. Pharmaceutical carriers for themicroRNA composition may or may not be included in the kit.

Although in some embodiments the kit comprises the microRNA composition,in other embodiments the microRNA composition is synthesized. Poly(A)polymerase may be from any source, but specifically contemplated is apoly(A) polymerase from yeast or E. coli, which may be recombinant orpurified from the organism. A reaction buffer for poly(A) polymerase maybe included in any kit of the invention. Typically, such a poly(A)polymerase reaction buffer includes a volume exclusion reagent, such asPEG, magnesium, and sodium. In certain embodiments, the poly(A)polymerase reaction buffer in the kit contains at least: about 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15% or more (or any range derivable therein)PEG; about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 mM or more MgCl₂ (orany range derivable therein); about 100, 200, 300, 400, 500, 600, 700,800, 900 mM NaCl (or any range derivable therein); about 50, 60, 70, 80,90, 100, 110, 120, 130, 140, 150 mM or more MES (or any range derivabletherein); and about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4 mM or more DTT (orany range derivable therein) The kits may also include a manganesesource, which may be included as a separate component of a kit or in asolution or buffer with other components, such as in the reaction bufferIt is contemplated that about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 mMor more of MnCl₂ is included m the kit

Nucleotides may also be included in kits of the invention. Nucleotidesmay be for DNA or RNA. Concentrations of a nucleotide or of a nucleotidemix (total concentration of all nucleotides) include, but are notlimited to, about, at least about, or at most about 0.5, 1.0, 1 5, 2 0,2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5 5, 6.0, 6.5, 7 0, 7.5, 8 0, 8 5, 90, 95, 10 0 mM or more (or any range derivable therein) Moreover, they maybe modified or not modified. If they are modified, they may have areactive group or they may have a label attached to it In certainembodiments, one or more nucleotides in a kit has a reactive group, suchas an amine-reactive group In other embodiments, a nucleotide is alreadylabeled. It may be labeled with a chemilummescent or fluorescent label,such as a dye. Specifically contemplated are amine-reactive dyes.Moreover, it is specifically contemplated that kits may or may notcontain both modified and unmodified nucleotides. Also, kits may containthe label that will be attached to the nucleotide. Any label that can beattached to a nucleotide, as well as any specifically identified herein,can be included in kits of the invention.

Individual components may also be provided in a kit in concentratedamounts; in some embodiments, a component is provided individually inthe same concentration as it would be in a solution with othercomponents. Concentrations of components may be provided as 1×, 2×, 5×,10×, or 20× or more.

For any kit embodiment, there can be nucleic acid molecules thatcomprise a sequence that is identical to all or part of SEQ ID NO:1, SEQID NO:2, or SEQ ID NO:3, or variants thereof. Any nucleic acid describedherein may be implemented as part of a kit.

The components of the kits may be packaged either in aqueous media or inlyophilized form.

The container means of the kits will generally include at least onevial, test tube, flask, bottle, syringe or other container means, intowhich a component may be placed, and preferably, suitably aliquoted.Where there is more than one component in the kit (labeling reagent andlabel may be packaged together), the kit also will generally contain asecond, third or other additional container into which the additionalcomponents may be separately placed However, various combinations ofcomponents may be comprised in a vial. The kits of the present inventionalso will typically include a means for containing the nucleic acids,and any other reagent containers in close confinement for commercialsale. Such containers may include injection or blow-molded plasticcontainers into which the desired vials are retained.

When the components of the kit are provided in one and/or more liquidsolutions, the liquid solution is an aqueous solution, with a sterileaqueous solution being particularly preferred.

However, the components of the kit may be provided as dried powder(s).When reagents and/or components are provided as a dry powder, the powdercan be reconstituted by the addition of a suitable solvent. It isenvisioned that the solvent may also be provided in another containermeans.

Such kits may also include components that facilitate isolation of thelabeled miRNA It may also include components that preserve or maintainthe miRNA or that protect against its degradation Such components may beRNAse-free or protect against RNAses. Such kits generally will comprise,in suitable means, distinct containers for each individual reagent orsolution.

A kit will also include instructions for employing the kit components aswell the use of any other reagent not included in the kit. Instructionsmay include variations that can be implemented

Kits of the invention may also include one or more of the following:Control RNA; nuclease-free water, RNase-free containers, such as 1.5 mltubes; RNase-free elution tubes, PEG or dextran, ethanol; acetic acid,sodium acetate; ammonium acetate; guanidimum, detergent; nucleic acidsize marker, RNase-free tube tips; and RNase or DNase inhibitors.

It is contemplated that such reagents are embodiments of kits of theinvention. Such kits, however, are not limited to the particular itemsidentified above and may include any reagent used for the manipulationor characterization of miRNA.

In some embodiments of the invention, additional anti-cancer agents areincluded in the kit. Examples include chemotherapeutics, hormone therapyagents, and immunotherapy agents.

EXAMPLES

The following examples are included to demonstrate some embodiments ofthe invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute some modesfor its practice. However, those of skill in the art should, in light ofthe present disclosure, appreciate that many changes can be made in thespecific embodiments which are disclosed and still obtain a like orsimilar result without departing from the spirit and scope of theinvention.

Example 1 Use of MIR-520G/H to Sensitize Epithelial Ovarian Cancers toChemotherapy

MicroRNAs (miRNAs) are small, non-coding RNA transcripts that play acritical role in silencing patterns of gene expression. To identifymicroRNAs useful as therapeutic targets for epithelial ovarian cancer,the genomic location of known human microRNAs was determined usingMirBase and correlated with genomic copy number variation identified ina dataset of >400 ovarian cancer specimens. In aspects of the invention,microRNAs whose patterns of gene expression are either tightly repressedor lost during the course of treatment for ovarian cancer are useful fortherapeutically targeting cancers, including ovarian cancers, forexample. The inventors identified a unique genomic locus at 19q13.41that encodes more than 48 individual microRNAs. Copy number losses atthis locus could be identified in ˜37% of high grade papillary serouscancers. Expression of individual 19q locus microRNAs did not appear tobe a prominent feature of reproductive tract tissues or cancers. Mimicsfor at least 3 of the microRNAs encoded by the 19q13.41 locus(mir-520a-3p, miR-520-5p, miR-520h) significantly impacted eitherproliferation or apoptosis in established ovarian cancer cell lines(SKOV4, HEY, OVCAR8, OVCAR5) when compared to control cultures. Ofthese, miR-520a and miR-520h decreased the IC₅₀ of multiple ovariancancer cell lines for cisplatin as much as 3-fold (p<0.01), regardlessof their impact on caspase activity. In examples of the invention,targets for miR-520a and miR-520h include >150 gene products previouslyimplicated, for example, in DNA damage pathways and ovarian cancer,including ATM, BRCA1, FANCD2, Bcl-2, AKT and E2F, for example. Inexemplary OVCAR8 cells, ectopic expression of miR-520h induced both thephosphorylation and degradation of ATM, targeted Caspase 7 and bluntedthe response of BRCA1 and other members of DNA damage repair pathways toincreasing concentrations of cisplatin.

Altered genomic copy number variation at the 19q13.41 microRNA locusoccurs frequently in cancers including epithelial ovarian cancers, andindividual microRNAs encoded by this locus are useful to sensitizeovarian cancers to platinum-based chemotherapy, for example. By routinemethods in the art, one can determine how these microRNAs exert theirbiologic effects, identify genetic signatures predictive of theirability to sensitize primary ovarian cancers to chemotherapy anddetermine how best and when to deliver specific miR520-a, g, and/or hmicroRNAs for optimal therapeutic impact.

Example 2 Exemplary Materials and Methods

Cell Culture and In Vitro Assays

Established ovarian cancer cell lines were obtained from the ATCC(OVCAR8, OVCARS) and the University of Texas M.D. Anderson Cancer Center(HEYA8, SKOV3ip1). All cell lines were maintained at 37° C. in RPMI 1640media (Invitrogen, Carlsbad, Calif.) supplemented with 10% fetal bovineserums (Hyclone, Logan, Utah) and 1% penicillin/streptomycin (Gibco,Carlsbad, Calif.). MicroRNA mimics and mimic controls (Dharmacon,Lafayette, Colo.) were transiently transfected into actively growingcultures using Lipofectamine 2000 (Invitrogen, Carlsbad, Calif.). Aftera 24-hour incubation, transfected cultures were washed, passaged andseeded into 96 well plates. Proliferation and apoptosis were measured at72, 96 and 120 hours after transfection using standard assays (CellTiter 96 Aqueous One; Caspase Glo 3/7; Promega, Madison, Wis.). pLemiRexpression vectors (Open Biosystems/Thermo, Huntsville, Ala.) wereprepared according to manufacturer's instructions and transfected intocells using Fugene 6 (Roche, Indianopolis, Ind.). Followingtransfection, stable clones were selected using puromycin (Invitrogen)at doses optimized for each line. Rates of proliferation and apoptosiswere examined in stable transfectants 24, 48, 72 and 96 hours afterselected cells had been passaged and dispensed into 96 well plates.Viability was assessed across a clinically relevant range of cisplatinconcentrations using an MTS assay (Promega). Cisplatin was obtained fromTeva Pharmaceuticals (Irvine, Calif.).

RNA Extraction and Quantitative RT-PCR

Total RNA was extracted from cell pellets using the mirVana miRNAextraction kit (Ambion, Austin, Tex.) according to the manufacturer'sinstructions. Levels of microRNA expression were examined using MatureMicroRNA Taqman assays (Applied Biosystems, Foster City, Calif.) usingU6 as a control. For all gene expression assays, cDNA was prepared usingqScript cDNA supermix (Quanta Biosciences, Gaithersburg, Md.). TaqMangene expression assays (Applied Biosystems) were used to measureexpression of specific target genes according to the manufacturer'sinstructions. All reactions were performed in triplicate usingOneStepPlus real time PCR system (Applied Biosystems). All qPCR resultsare reported using the ΔΔC_(T) method (17).

Western Blot Analysis

Whole cell lysates equivalent to 30 μg of protein were resolved on 4-12%gradient SDS-PAGE. Resolved proteins were transferred onto PVDFmembranes using the iBlot system (Invitrogen). Rabbit polyclonal and/ormouse monoclonal antibodies specifically recognizing ATM, phospo-ATM(Ser1981) CHK1 phospho-CHK1 (Ser216), p21, phospo-CHK2 (Tyr68), BCL2,XIAP, CDC25A were obtained from Cell Signaling Technologies (Danvers,Mass.). A rabbit polyclonal antibody recognizing FANCD2 was alsopurchased from Abcam (Cambridge, Mass.). Protein expression wasvisualized using species-specific, horseradish peroxidase-conjugatedsecondary antibody (Cell Signaling) and ECL Plus chemiluminescentsubstrate (Amersham, Buckinghamshire, UK).

In Vivo Experiments

Permission to perform animal experiments was obtained from theInstitutional Animal Care and Use Committee (IACUC) for Baylor Collegeof Medicine. Eight-week female athymic (Foxn1^(nu/nu)) mice werepurchased from the National Cancer Institute-Charles River andmaintained on a standard diet ad lib. Each animal was inoculatedintraperitoneally with 2.5×10⁶ cells stably transfected with vectordriving miR-520h, a non-targeting miRNA control or empty vector.Expression of miR-520h was measured in cell lines using Taqman assaysimmediately prior to inoculation. Tumor xenografts were followed weeklyfor tumor progression or regression using the Image Station In-Vivo FXsystem (Kodak Molecular Imaging Systems, New Haven, Conn.) usingendogenous RFP encoded by the pLemiR vector as a tag. At each timepoint, mice were anesthetized with isoflurane and placed in an imagingchamber, after which, fluorescent intensity images with x-ray overlaywere obtained and analyzed. At the completion of the study, mice wereeuthanized after sedation under isoflurane (Abbott Laboratories, NorthChicago, Ill.). For experiments involving treatment, cisplatin wasadministered at 5 mg/kg to mice by tail vein injection weekly for 4weeks, after which, 1.5 mg/kg was administered biweekly. Tumor volumeand distribution for all animals in each experiment were documented andcorrelated with imaging results.

Example 3 Exemplary Results

To identify microRNAs potentially useful as therapeutic targets inovarian cancer, the inventors screened known human microRNAs (v15.0) asa function of the copy number variation observed at their genomic lociin a dataset of 500 epithelial ovarian cancers (EOC). Using this data,the inventors identified specific microRNAs that sensitize human cancersto cytotoxic chemotherapy, including miR-520a/g/h. Copy number losses atthe genomic locus encoding these miRNAs could be detected in ˜37% ofovarian cancer specimens, whereas copy number gains were observed in˜35%. In general, low levels of expression for the individual microRNAsencoded by this locus were observed when the same set of ovarian cancerspecimens were screened using a custom miRNA expression array,indicating that levels of the microRNAs encoded by this locus aretypically repressed. To more closely examine this question, theinventors interrogated a comprehensive database of microRNA transcriptspreviously compiled from reproductive tract tissues and ovarian cancersstudied using Next Generation Sequencing (NGS). Using this dataset,there was only rare evidence for expression of miR-520a/g/h microRNAs ineither distal fallopian tube (n=14) or primary cultures of normalovarian surface epithelia (n=7). In addition, very few transcriptscorresponding to mature miR-520a/g/h microRNAs were identified inepithelial ovarian cancers, regardless of whether papillary serous(n=12), endometrioid (n=6) or clear cell cancers (n=5) were examined.Collectively, these observations indicate that expression ofmiR-520a/g/h microRNAs are tightly repressed in normal femalereproductive tract tissues and that dysregulated expression of 19q13.41microRNAs occurs, but is not a predominant feature of epithelial ovariancancers.

To assess the biologic impact of dysregulated 19q locus microRNAexpression in those ovarian cancers where they are expressed, theinventors parsed groups of ovarian cancer patients according to levelsof 19q cluster microRNAs in a stepwise fashion using data recentlygenerated by the TCGA. Kaplan-Meier analysis was used to comparesurvival for each group determined in this fashion and define groups ofovarian cancer patients where differences in microRNA expression hadtheir most robust association with outcome demographics. Using thisapproach, there was a strong correlation between levels of miR-520g/hexpression and improved survival when the group of 240 patients withlowest miR-520g/h expression were compared to outcomes for the 120patients with highest levels of miR-520g/h expression (Z-score of 2.7,p<0.001). These observations indicate that dysregulated expression ofmiR-520h favorably impacts ovarian cancer outcomes in a specific subsetof patients in which levels are expressed beyond an arbitrarily definedthreshold.

Expression of individual 19q13.41 locus microRNAs acutely impact ovariancancer growth and apoptosis.

To explore 19q13.41 locus microRNAs as being useful to therapeuticallytarget ovarian cancer, the inventors transfected mimics for miR-520a/g/hmicroRNAs into established ovarian cancer cell lines. The expression ofdifferent microRNAs encoded by the 19q13.41 genomic locus individuallyimpact the rates of proliferation or apoptosis in these cells. Asdemonstrated in FIGS. 2A-2H, mimics for miR-520h increased theproliferation of OVCAR8 cells by an average of 19% (n=6, p<0.001) whencompared to non-silencing control 96 hours following transfection (FIG.2E). However, short-term transfections with mimics for miR-520h had noimpact on apoptosis. In contrast, miR-520a-3p and miR-520a-5p decreasedapoptosis in OVCAR8 cells by 16%, 20% and 22% respectively, whencompared to controls (FIGS. 2A-2H, n=6 each microRNA, p<0.002). Inscreening additional cell lines, mimics for miR-520h were found toincrease rates of apoptosis by (n=6, p<0.01) when acutely transfectedinto HEYA8 cells with no significant impact on proliferation.Transfection of miR-520h mimics had no impact on proliferation orapoptosis in either OVCAR5 (n=6) or HEY cells (n=6). Mimics for at leastmiR-520a and miR-519c impacted proliferation and/or apoptosis in somebut not all ovarian cancer cell lines tested.

MiR-520g/h Sensitizes Established Ovarian Cancer Cell Lines toPlatinum-Based Chemotherapy

Recent evidence suggests that microRNA functions may only become evidentunder conditions of biologic stress. For this reason, the inventorscreated clones of OVCAR8 and SKOV3ip1 ovarian cancer cell lines stablyexpressing either miR-520h (n=3) and miR-520a (n=3). Similar to resultswith short term transfections, stable expression of miR-520h enhancedthe proliferation of both OVCAR8 and SKOV3ip1 cells and inhibitedexpression caspase 3/7 expression. To assess the impact of thesemicroRNAs on cisplatin-induced cell killing, the inventors examined theviability of OVCAR8 and SKOV3ip1 cells stably expressing either microRNAincubated with clinically relevant concentrations of cisplatin. Stableexpression of miR-520h significantly sensitized both OVCAR8 and SKOV3ip1cells to cisplatinum (FIGS. 4C, 4D). The IC₅₀ for cisplatinum in OVCAR8cells stably expressing miR-520h was 0.54 μg/ml compared to 1.31 μg/mlin OVCAR8 cells expressing a non-targeting mimic control. The IC₅₀ forcisplatin in OVCAR8 cells expressing miR-520a was found to be 0.867μg/ml. For SKOV3ip1 cells, the presence of miR-520h decreased IC₅₀ to0.298 μg/ml as compared to 0.429 μg/ml for cells expressing miR-520a and0.43 μg/ml for cells expressing the non-silencing control. It should benoted that SKOV3ip1 cells tolerated only very low levels of miR-520aexpression. The impact of cisplatin treatment on rates of apoptosisdiffered dramatically between the two cell lines. In p53 wildtypeSKOV3ip1 cells, significantly increased rates of apoptosis were observedin the presence of either miR-520g/h or miR-520a (FIGS. 4C, 4D; n=12,p<0.001 at all time points tested). However, in p53-mutated OVCAR8cells, significantly decreased levels of apoptosis were observed.

ATM is a Target for miR-520g/h Mediated Gene Silencing in OvarianCancers.

To better understand the mechanisms by which miR-520g/h impacts ovariancancer growth, apoptosis and/or metastasis, the inventors usedestablished bioinformatic algorithms (Targetscan, MiRanda) to identifyits potential targets in ovarian cancer. They also examined the functionof these putative targets using pathway analysis tools including Dianaand Ingenuity software. miR-520h was predicted to target more than 3,841individual genes, including a large number of key oncogenes previouslyknown to be critical for the ongoing progression and/or metastasis ofepithelial ovarian cancer. These included FANCD2, ATM, BRCA1, ERα, ERβ,PR, p21/WAF1, FAS, Caspase 7, Cyclin B, Cyclin D, Wee 1, XIAP and bcl2.Many of these gene products have been previously implicated in ovariancancer and are known to play important roles in critical cell functions,including regulation of the G1-S and G2-M cell cycle checkpoints,apoptosis and DNA damage repair pathways. An exemplary model summarizingseveral findings can be found in FIG. 7.

The bioinformatic analyses indicated that ATM is a critical targetmediating the biologic effects of miR-520h in ovarian cancer. Todetermine whether increased expression of miR-520h targeted ATM fortranslational silencing, the inventors examined levels of ATM expressionin OVCAR8 cells expressing mimics for miR-520g/h. Expression of ATMprotein was significantly less in OVCAR8 cells expressing miR-520g/hthan control cultures. (FIG. 8A). However, levels of ATM transcript werefound to be paradoxically higher in cells expressing miR-520h thannon-silencing control when compared by qPCR (FIG. 8C). In addition, theexpression of miR-520g/h led to increased levels of an ATM cleavageproduct (ΔATM) in a dose-dependent fashion when OVCAR8 cells wereincubated with increasing concentrations of cisplatin (FIG. 8B; n=3).Generation of this cleavage product has been previously linked to theactivation of caspases, including caspase 7, that promote ATMdegradation and inactivation. Of note, the relative expression of ATMtranscripts in OVCAR8 cells is actually higher in presence of miR-520g/hthan non-silencing miRNA control (FIG. 8C). This indicates that thestable expression of miR-520g/h has resulted in increased levels of ATMmRNA at baseline in the absence of cisplatin chemotherapy. Presence ofmir-520g/h continues to suppress ATM expression at the protein level. Insome embodiments this occurs as the direct result of translationinhibition by the miR-520g/h microRNA or in alternative embodiments thisis an indirect result of altered caspase expression observed at baselinein these cells (FIG. 8C).

The inventors also examined a number of gene products whose functionand/or expression are either predicted by an algorithm to be directlytargeted by miR-520h or whose function might be regulated by alteredlevels of ATM activity. In general, increased expression of miR-520g/hresults in decreased levels of the phosphorylated, active form of ATM,consistent with decreased levels of its expression in OVCAR8 cellsstably expressing miR-520g/h as well as potentiated degradation of ATMobserved when these cells were treated with platinum. However, increasedATM activity does not appear to influence all downstream mediatorsregulated by this gene product similarly. ATM has been previously shownto regulate CHK1 function by driving its phosphorylation. However,levels of CDC25c expression are much lower in the miR-520g/h expressingcells, particularly when exposed to cisplatin. This finding isexplained, in certain embodiments, by the observation thatplatinum-induced expression of CHK1 is inhibited by miR-520h, likely bydirect targeting of the CHK1 transcript for degradation (FIG. 8D).Expression of miR-520g/h blunts the response of additional gene productspredicted implicated in DNA damage repair pathways and apoptosis whencells are exposed to cisplatin. As demonstrated in FIG. 8, these geneproducts include BRCA1 and CASPASE 7. Each of these latter two geneproducts is predicted to be directly targeted by miR-520g/h. Theirresponses to cisplatin treatment are significantly less in OVCAR8 clonesstably expressing miR-520g/h than clones expressing non-silencingcontrol.

In embodiments of the invention, miR-520g/h works to create a syntheticlethal phenotype in ovarian cancer cells by targeting multiple DNAdamage repair pathways. The ability of miR-520g/h to target BRCA1 is animportant example of this.

MiR-520g/h Impacts Ovarian Cancer Growth and Metastatis In Vivo

The in vitro observations indicate that miR520g/h and/or miR-520a alterthe proliferations of established ovarian cancer cell lines and in someembodiments are reasonably expected to promote their growth andmetastasis in vivo. The inventors utilized xenograft models tocharacterize this embodiment. Foxn1^(nu/nu) mice were implantedsubcutaneously or intraperitoneally with 2.5 million cells stablytransfected with miR-520g/h, non-silencing microRNA control or emptyvector alone. All animals were then followed, imaging the growth andmetastasis of xenografts cells using RFP stably expressed by the ovariancancer cell line. As demonstrated in FIGS. 5A-E, a larger number ofsmaller xenografts are generated by the intraperitoneal inoculation ofOVCAR8 cells stably expressing miR-520g/h than control cells stablytransfected with non-coding vector or empty vector alone (n=3, p<0.008).This difference was documented not only when tumor burden was measuredaccording to the number and size of RFP active tumors that met imagingthresholds, but could also directly measured at necropsy (FIG. 5F).Total intraperitoneal tumor burden between the three groups did notdiffer significantly, indicating that ectopic expression of miR-520haltered the pattern of cancer metastasis in vivo. Consistent with thisconsideration, a second experiment was performed where ovarian cancercells expression miR-520h were compared to cells expressing either anon-targeting miRNA control or empty vector alone. Results of thisexperiment demonstrate that implant size total tumor burden wassignificantly smaller in xenografts created by subcutaneous inoculationin a manner that produces a single infiltrative tumor nodule (FIG. 5G).

MiR-520h Improves Survival for Animals Xenografted with Ovarian CancerCells

To determine whether expression of miR-520h impacted responses ofovarian cancer xenografts to systemically administered chemotherapy, theinventors inoculated Fox1nu/nu mice with 4×10⁶ OVCAR8 cells stablyexpressing either miR-520h mimic, a non-targeting mimic control or emptyvector. Once tumor implants were readily palpable, weekly administrationof cisplatin equivalent to a dose of 5 mg/kg was initiated and givenweekly for a total of 4 weeks. This course of treatment was specificallyused to mimic the typical course experienced by women undergoingtreatment for this illness. Xenografts in experimental groups respondedto this treatment regimen with a nearly complete elimination of diseasein animal inoculated with OVCAR8 cells stably expressing miR-520h (FIGS.6A-6F), whereas the low doses of cisplatin used in these experiments hadmuch less impact on shrinking the tumor burden in control implants. Thisobservation is consistent with a sensitization of ovarian cancer cellsto cisplatin by miR-520h mimic. Cisplatin was continued approximatelybiweekly at a reduced dose of 1.5 mg/kg until disease progressionrequiring euthanasia of animals by IACUC criteria. As demonstrated inFIGS. 6A-6G, the mean survival for animals xenografted with OVCAR8 cellsstably expressing mir-520h was 114 days, an increase of ˜44% whencompared to control animals inoculated OVCAR8 cells stably expressingempty vector (FIG. 6G). In contrast, median survival for animalsxenografted with OVCAR8 cells stably transfected with empty vector orOVCAR8 cells stably expressing non-targeting microRNA control was 79days and 90 days, respectively. At necropsy, examination of theseanimals demonstrated that implants were significantly smaller in animalsxenografted with OVCAR8 cells stably expressing miR-520h compared toeither non-targeting mimic control or empty vector.

Mimics for miR-520g/h impact creation of spheroids, a key intermediatein metastasis and cancer stem cells and impact patterns of cancerstemness. OVCAR8 and SVOK3ip1 cells stably expressing mimics formiR-520h demonstrate decreased capacity to form spheroids, multicellularaggregates that play a key role in ovarian cancer metastasis. (FIG. 9A)Spheroids have also been used to isolate subpopulations of cells thatfulfill many of the criteria of cancer stem cells and express enhancedlevels of specific gene products including EZH2, OCT4 and NANOGassociated with pleuripotency in human embryonic stem cells (16). (FIG.9B) Expression of miR-520gh impacts patterns of and eliminates increasedexpression of specific patterns of gene expression (i.e. lin28A/B)associated with stemness that are induced when ovarian cancer cells(OVCAR8 and SKOV3ip 1) are cultured in media that induce spheroidformation.

Example 4 MIR-520H Sensitizes Cell Lines Derived from Uterine Cancer toCisplatin

FIG. 10 demonstrates that miR-520h sensitizes cell lines derived fromuterine cancer to cisplatin. UPSC Ark1 cells stably transfected withlentiviral vector driving the expression of either miR-520h or anon-targeting miRNA control were incubated with increasingconcentrations of cisplatin. As demonstrated in FIG. 10, the IC50 forcisplatin was reduced by more than 90% in cells expressing miR-520h.

Example 5 Significance of Embodiments of the Invention

Understanding molecular events critical for sensitizing ovarian cancersto the established cell-killing effects of platinum-based chemotherapyis an important goal not only for determining how best to initiallytreat women newly diagnosed with this disease but also reducing thetoxicity associated with the use of platinum-based chemotherapy andpotentially reversing the resistance that emerges with continue platinumtreatment. Unfortunately, the molecular mechanisms responsible forplatinum resistance in ovarian cancer remain poorly understood.Recently, a number of investigators have begun to examine this questionat a molecular level, identifying multiple gene products potentiallyinvolved in the emergence of platinum resistance. Their efforts havepointed to a number of cell pathways, including the TGF-β signalingpathway as well as DNA damage repair pathways as playing a critical rolein determining whether ovarian cancers respond to platinum-basedchemotherapy (17-19, 21). Particularly interesting are observations thatplatinum-sensitivity is at least partly determined by patterns of geneexpression that have been associated with mutations in the ovariancancer susceptibility gene BRCA1 (22).

The role of microRNAs in determining the sensitivity of ovarian cancersto chemotherapy remains largely unexplored. The inventors screened knownhuman microRNAs with the goal of identifying individual microRNAspotentially suitable for this goal. In certain aspects of the invention,microRNAs that remain silencing or whose expression may be lost or whoselevels of expression are suppressed during the course of platinum-basedtreatment are useful targets for sensitizing ovarian cancers tochemotherapy. The inventors identified miRNA 520-a, g, and/or h that incertain embodiments of the invention undergo frequent copy number gainsand loss in ovarian cancers. Despite this, levels of expression for theindividual microRNAs encoded by this locus are typically quite low ornot expressed. However, the data clearly establish that the ectopicexpression of at least one exemplary microRNA, miR-520h, can be used toimpact the biologic behavior of ovarian cancer both in vitro and invivo, despite the fact that it is not expressed in ovarian cancer celllines in which it has been tested. In part, miR-520h accomplishes thisfeat via the translational inhibition of ATM expression and increasingthe rate at which this tumor suppressor is degraded, in certainembodiments. Other investigators have demonstrated that the generationof ΔATM in ovarian and other types of cancer cells are primarily due toCaspase 7 activity (23,24). However, it is not presently clear whetherthis also holds true for ovarian cancer cells treated with miR-520h asthe results also indicate that Caspase 7 is a target for MiR-520hmediated gene silencing and that its expression to cisplatin treatmentare blunted by the presence of miR-520h. In addition, in someembodiments miR-520h contributes to the sensitization of ovarian cancersby also targeting the expression of multiple, other oncogenes and tumorsuppressors implicated in ovarian cancer and whose function has beenshown to be involved not only in apoptosis, but also DNA damage repair.

The data also provide insight into the mechanisms by which ovariancancers can be sensitized to cisplatin. miR-520h sensitize bothp53-mutated OVCAR8 cells and p53-null SKOV3ip1 cells. However, miR-520happears to sensitize p53-null SKOV3ip1 cells to a much greater degreethan p53-mutated OVCAR8 cells. In addition, the inventors see verydifferent apoptotic responses in these two cell lines in response toplatinum treatment in the presence and absence of miR-520h. Thedifferences in the rates of apoptosis observed in response to cisplatinin OVCAR8 and SKOV3ip1 cells are not necessarily surprising given thatthese two cells lines have very different patterns of p53 expression,for example. In specific embodiments, differences in apoptotic responsein OVCAR8 and SKOV31ip1 cells are because of the differences in the p53status of these two different ovarian cancer cell lines.

The observations have identified a number of individual microRNAs usefulas therapeutic targets in at least ovarian cancer. The resultscontribute significantly to understanding of whether or how microRNAscan be used for therapeutic purposes. These observations indicate thatthe mechanisms are susceptible in a novel approach differently from howmany investigators are currently considering the use of microRNAs astherapeutic targets in human disease.

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All patents and publications mentioned in the specification areindicative of the level of those skilled in the art to which theinvention pertains. All patents and publications are herein incorporatedby reference in their entirety to the same extent as if each individualpublication was specifically and individually indicated to beincorporated by reference.

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5. Sehouli J, Stengel D, Harter P, Kurzeder C, Belau A, Bogenrieder T,et al. Topotecan Weekly Versus Conventional 5-Day Schedule in PatientsWith Platinum-Resistant Ovarian Cancer: a randomized multicenter phaseII trial of the North-Eastern German Society of Gynecological OncologyOvarian Cancer Study Group. Journal of clinical oncology: officialjournal of the American Society of Clinical Oncology. 2011; 29:242-8.

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Although the present invention and its advantages have been described indetail, it should be understood that various changes, substitutions andalterations can be made herein without departing from the spirit andscope of the invention as defined by the appended claims. Moreover, thescope of the present application is not intended to be limited to theparticular embodiments of the process, machine, manufacture, compositionof matter, means, methods and steps described in the specification. Asone of ordinary skill in the art will readily appreciate from thedisclosure of the present invention, processes, machines, manufacture,compositions of matter, means, methods, or steps, presently existing orlater to be developed that perform substantially the same function orachieve substantially the same result as the corresponding embodimentsdescribed herein may be utilized according to the present invention.Accordingly, the appended claims are intended to include within theirscope such processes, machines, manufacture, compositions of matter,means, methods, or steps.

What is claimed is:
 1. A method of sensitizing uterine cancer in anindividual to a cancer treatment that is platinum-based chemotherapy,comprising the step of administering to the individual an effectiveamount of a composition as follows: a RNA polynucleotide comprising SEQID NO:1, SEQ ID NO:2, or SEQ ID NO:3, wherein upon administering thecomposition to the individual, the uterine cancer is thereby sensitizedto the platinum-based chemotherapy treatment.
 2. The method of claim 1,wherein the composition is delivered to the individual by liposome,nanosphere, nanoparticle, nanodiamonds, impregnanted polymer, multistagenanoparticles and/or gels.
 3. The method of claim 1, wherein thecomposition is delivered to the individual intravenously orintraperitoneally.
 4. The method of claim 1, wherein the composition isdelivered to the individual prior to the cancer treatment.
 5. The methodof claim 1, wherein the composition is delivered to the individualsubsequent to the cancer treatment.
 6. The method of claim 1, whereinthe composition is delivered to the individual concomitantly with thecancer treatment.